Cyclic maltosylmaltose, cyclic maltosymaltose-forming enzyme, their preparation and uses

ABSTRACT

An object of the present invention is to provide an option of non-reducing saccharide by providing a novel non-reducing saccharide composed of glucose as constituents and to provide a novel enzyme forming the non-reducing saccharide, a method and process for producing the same, a DNA encoding the enzyme, a recombinant DNA and transformant comprising the DNA, a composition comprising the non-reducing saccharide, and uses thereof. The present invention solves the above object by providing a novel cyclic saccharide having a structure of cyclo{&gt;6)-α-D-glucopyranosyl-(1&gt;4)-α-D-glucopyranosyl-(1&gt;6)-α-D-glucopyranosyl-(1&gt;4)-α-D-glucopyranosyl-(1&gt;}, cyclic maltosylmaltose, novel cyclic maltosylmaltose-forming enzyme, a method and process for producing the same, a DNA encoding the enzyme, a recombinant DNA and transformant comprising the DNA, a composition comprising the cyclic maltosylmaltose or a saccharide composition comprising the same, and uses thereof.

The present application is a divisional application of U.S. applicationSer. No. 10/569,959, filed Feb. 28, 2006, which is a U.S. National Stageof PCT/JP04/12282 filed Aug. 26, 2004; now allowed, the entire contentsof each and both these applications being hereby incorporated byreference herein.

TECHNICAL FIELD

The present invention relates to a cyclic maltosylmaltose having astructure ofcyclo{>6)-α-D-glucopyranosyl-1>4)-α-D-glucopyranosyl-(1>6)-α-D-glucopyranosyl-(1>4)-α-D-glucopyranosyl-(1>}(hereinafter, may be simply abbreviated as “cyclic maltosylmaltose” or“CMM” in this specification), CMM-forming enzyme, their preparation anduses; and a DNA encoding the enzyme and a recombinant DNA comprising theDNA. More particularly, the present invention relates to CMM,CMM-forming enzyme, their preparation, a microorganism producing theenzyme, a DNA encoding the enzyme, a recombinant DNA comprising the DNA,a transformant, a method and process for producing CMM by using theenzyme, and a composition comprising CMM.

BACKGROUND ART

There have been known saccharides composed of glucose molecules asconstituents, for example, partial hydrolyzates, produced from starchesas materials, including amyloses, amylodextrins, maltodextrins,maltooligosaccharides, and isomaltooligosaccharides. Also, thesesaccharides are known to have usually non-reducing ends and reducinggroups at their molecular ends and to exhibit reducing power. Usually,reducing power on a dry solid basis of partial starch hydrolyzates isrepresented by Dextrose Equivalent (DE). Partial starch hydrolyzateswith high DE values are known to have a relatively low molecular weight,relatively low viscosity, strong sweetness and reactivity, easyreactivity with amino group-containing substances such as amino acidsand proteins by amino carbonyl reaction that may induce browning andunpleasant smell and easily cause deterioration. In order to improvethose disadvantages, methods for decreasing or eliminating the reducingpower of partial starch hydrolyzates without altering glucose residueshave been required for a long time. “Journal of American ChemicalSociety, Vol. 71, 353-358 (1949)” discloses a method to produce α-, β-or γ-cyclodextrin, constructed by 6, 7 or 8 glucose molecules bound bythe α-1,4 glucosidic linkage, from starch by “macerans amylase”. Atpresent, these cyclodextrins are produced on an industrial scale and areapplied to various uses because of their non-reducing power,tastelessness, and clathrating abilities. Further, Japanese Patent KokaiNos. 143,876/95 and 213,283, applied for by the same applicant as thepresent invention, discloses a method to convert maltooligosaccharidesand partial starch hydrolyzates into trehalose, composed of two glucosemolecules linked together via the α,α-1,1 linkage, by contacting anon-reducing saccharide-forming enzyme and a trehalose-releasing enzyme.At present, trehalose is produced from starch on an industrial scale andis applied to various uses because of its non-reducing power and itsmild and high quality sweetness. While, International Patent ApplicationNos. WO 01/90338 A1, WO 02/055708 A1, and WO 02/40659 A1, applied for bythe same applicant as the present invention, disclose a method toproduce acyclic tetrasaccharide, having a structure of binding fourglucose molecules via alternating α-1,3 and α-1,6 glucosidic linkages,i.e.,cyclo{>6)-α-D-glucopyranosyl-(1>3)-α-D-glucopyranosyl-(1>6)-α-D-glucopyranosyl-(1>3)-α-D-glucopyranosyl-(1>},from starch or partial starch hydrolyzates by contactingα-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme. The cyclic tetrasaccharide hasabilities of clathrating other substances because of its cyclicstructure and stabilizing volatile organic substances. Further, sincethe saccharide has no reducing power, it is expected that the saccharidecan be used and processed without causing browning and deterioration byamino-carbonyl reaction.

As described above, α-, β- or γ-cyclodextrin having a glucosepolymerization degree of 6, 7 or 8, trehalose having a glucosepolymerization degree of 2, and cyclic tetrasaccharide having astructure ofcyclo{>6)-α-D-glucopyranosyl-(1>3)-α-D-glucopyranosyl-1>6)-α-D-glucopyranosyl-(1>3)-α-D-glucopyranosyl-(1>},are used in various fields on the basis of these respective advantage asnon-reducing saccharides composed with glucose molecules. While, ifother non-reducing saccharides distinct from the above saccharides wouldbe provided, we would have more choice of using non-reducingsaccharides, and application thereof on various uses can be expected.

DISCLOSURE OF INVENTION

An object of the present invention is to provide an option ofnon-reducing saccharide by providing a novel non-reducing saccharidecomposed of glucoses as constituents and to provide a novel enzymeforming the non-reducing saccharide, a method and process for producingthem, a DNA encoding the enzyme, a recombinant DNA and transformantcomprising the DNA, a composition comprising the non-reducingsaccharide, and uses thereof.

To solve the above object, the present inventors have extensivelyscreened microorganisms capable of producing a novel non-reducingsaccharide-forming enzyme which forms a novel non-reducing saccharidewhen allowed to act on partial starch hydrolyzates. As a result, thepresent inventors isolated a novel microorganism of the genusArthrobacter, named “M6”, from a soil in Okayama-city, Okayama, Japan,and found that the microorganism produces a novel enzyme which forms aremarkable amount of a novel cyclic saccharide having a structure ofcyclo{>6)-α-D-glucopyranosyl-(1>4)-α-D-glucopyranosyl-(1>6)-α-D-glucopyranosyl-1>4)-α-D-glucopyranosyl-(1>},i.e. CMM, when allowed to act on α-1,4 glucans such as starches andpartial hydrolyzates. The present inventors also revealed the propertiesof CMM-forming enzyme and established the process for producing theenzyme. The present inventors also established a DNA encoding theenzyme, a recombinant DNA comprising the DNA, a transformant, a methodfor forming CMM by the enzyme, and processes for producing CMM and asaccharide composition comprising the same by using the enzyme. Also, itwas found that CMM can be easily collected by crystallizing CMM from itssupersaturated aqueous solution. Further, it was found that CMM hasuseful characteristics of clathrating volatile substances such asmethylalcohol, ethylalcohol, and acetic acid; showing no browning anddeterioration by amino carbonyl reaction; having a satisfactorystability to heating and the change of pH; and having a lowdigestibility and low fermentability. Furthermore, it was found that acomposition, comprising CMM or a saccharide composition comprising thesame, for example, foods and beverages with a high quality and asatisfactory flavor, low calorie foods and dietary foods, cosmetics withhigh quality and stability, pharmaceuticals with high activity andstability, etc., can be easily produced. The present invention wasaccomplished based on the above knowledge.

The present invention solves the above object by providing a novelcyclic saccharide having a structure ofcyclo{>6)-α-D-glucopyranosyl-(1>4)-α-D-glucopyranosyl-(1>6)-α-D-glucopyranosyl-(1>4)-α-D-glucopyranosyl-(1>},i.e. cyclic maltosylmaltose, a novel cyclic maltosylmaltose-formingenzyme, a method and process for producing them, a DNA encoding theenzyme, a recombinant DNA and transformant comprising the DNA, acomposition comprising the cyclic maltosylmaltose or a saccharidecomposition comprising the same, and uses thereof.

According to the present invention, an option of non-reducing saccharidecomposed of glucose as constituents can be extended. Further, thepresent invention enables the provision of CMM, a novel cyclicsaccharide which has been ever unknown, in large scale and the use ofCMM in a various fields including foods and beverages, cosmetics, andpharmaceuticals.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the HPLC elution pattern of the preparation of non-reducingsaccharide.

FIG. 2 shows the ¹H-NMR spectrum of the isolated non-reducingsaccharide.

FIG. 3 shows the ¹³C-NMR spectrum of the isolated non-reducingsaccharide.

FIG. 4 shows the structure of CMM of the present invention.

FIG. 5 shows the optimum temperature of CMM-forming enzyme.

FIG. 6 shows the optimum pH of CMM-forming enzyme.

FIG. 7 shows the thermal stability of CMM-forming enzyme.

FIG. 8 shows the pH stability of CMM-forming enzyme.

FIG. 9 shows a recombinant DNA, pBMB1, of the present invention.

In the figure, a section indicated with black bold line is a DNAencoding CMM-forming enzyme of the present invention, derived fromArthrobacter globiformis M6 (FERM BP-8448).

FIG. 10 shows the powdery X-ray diffraction pattern of crystalline CMM.

FIG. 11 shows the thermogravimetric curve of crystalline CMM.

EXPLANATION OF SYMBOLS

-   -   a: Glucose residue bound via the α-1,4 glucosidic linkage by        hydroxyl group at C-1 position    -   b: Glucose residue bound via the α-1,6 glucosidic linkage by        hydroxyl group at C-1 position    -   f1 (+) ori: Replication origin of f1 phage    -   Amp: Ampicillin resistance gene    -   Col E1 ori: Replication origin of colicin E1

BEST MODE FOR CARRYING OUT THE INVENTION

Cyclic maltosylmaltose (CMM) as referred to as in the present inventionmeans a cyclic tetrasaccharide where four glucose molecules are boundvia alternating α-1,4 and α-1,6 glucosidic linkages, i.e. a cyclictetrasaccharide having a structure ofcyclo{>6)-α-D-glucopyranosyl-(1>4)-α-D-glucopyranosyl-(1>6)-α-D-glucopyranosyl-(1>4)-α-D-glucopyranosyl-(1>}.The saccharide is a novel and ever unknown saccharide, firstly found ina culture medium of a microorganism isolated from a soil by the presentinventors. The present invention encompasses a cyclic tetrasaccharideconstructed by glucose without being restricted by its source, form,purity, and process for producing, as far as it has the above mentionedstructure.

CMM-forming enzyme as referred to as in the present invention means anyenzyme which acts on α-1,4 glucans having a glucose polymerizationdegree of 3 or higher to form 6-α-maltosyl-α-1,4-glucan by transferringmaltose to the C-6 position of glucose residue at the non-reducing endof other α-1,4 glucan; and successively forms CMM having a structure ofcyclo{>6)-α-D-glucopyranosyl-(1>4)-α-glucopyranosyl-1>6)-α-D-glucopyranosyl-(1>4)-α-D-glucopyranosyl-(1>}by a cyclization reaction. CMM-forming enzyme of the present inventionencompasses any enzyme catalyzing the above reaction without beingrestricted by its source, form, and purity (crude or purified).

The enzyme activity of CMM-forming enzyme of the present invention canbe assayed as follows: A substrate solution is prepared by dissolvingsoluble starch in 50 mM acetate buffer (pH 6.0) containing 2 mM CaCl₂ togive a concentration of 2% (w/v). A half milliliter of an enzymesolution is added to 0.5 ml of the substrate solution, and the mixturesolution is incubated at 40° C. for 30 min. After stopping the reactionby heating at about 100° C. for 10 min, the reaction mixture is admixedwith 4,000 units/g-solid of α-glucosidase and 250 units/g-solid ofglucoamylase to hydrolyze remaining soluble starch and by-products,oligosaccharides, and followed by the enzyme treatment at 50° C. for 1hour. The amount of CMM contained in the treated mixture is determinedby HPLC described later in Experiment 1. One unit activity ofCMM-forming enzyme is defined as the amount of enzyme which forms oneμmole of CMM per minute under the above conditions.

As a concrete example of CMM-forming enzyme, the enzyme having thefollowing physicochemical properties can be used.

(1) Molecular Weight

-   -   72,000±20,000 daltons when determined on SDS-PAGE;

(2) Isoelectric Point

-   -   pI 3.6±0.5 on isoelectrofocusing using ampholine, a carrier        ampholyte;

(3) Optimum Temperature

-   -   50 to 55° C. when reacted at pH 6.0 for 30 min;

(4) Optimum pH

-   -   pH 5.5 to 6.0 when reacted at 40° C. for 30 min;

(5) Thermal Stability

-   -   Stable up to 30° C. when incubated at pH 6.0 for 60 min    -   Stable up to 50° C. in the presence of 1 mM Ca²⁺ ion; and

(6) pH Stability

-   -   Stable in a pH range of 5.0 to 9.0 when incubated at 4° C. for        24 hours;

As an example of CMM-forming enzyme of the present invention, having theabove physicochemical properties, may have an amino acid sequence of SEQID NO:1 as the N-terminal amino acid sequence.

Usually, CMM-forming enzyme of the present invention has a prescribedamino acid sequence. For example, an amino acid sequence of SEQ ID NO:2or that homologous to SEQ ID NO:2 ca be listed. A variant enzyme havingan amino acid sequence homologous to SEQ ID NO:2 means an enzyme havingan amino acid sequence where one or more amino acids in SEQ ID NO:2 aredeleted, replaced or added without altering the enzyme activity ofacting on α-1,4 glucan having a glucose polymerization degree of 3 orhigher and producing CMM. As such a variant enzyme, it is preferablethat the enzyme has an amino acid sequence with a homology to SEQ IDNO:2 of, usually, 60% or higher, desirably, 70% or higher, moredesirably, 80% or higher, most desirably, 90% or higher.

However, CMM-forming enzyme, having the physicochemical properties orthe amino acid sequence described above, is just an example. CMM-formingenzyme of the present invention includes any enzyme having differentphysicochemical properties or amino acid sequences from the above ones,as long as it produces CMM.

Although CMM-forming enzyme of the present invention is not restrictedby its source, bacteria, particularly, the bacterial strain M6 isolatedfrom a soil by the present inventors can be preferably used as thesource. The following are the identification results of the strain M6capable of producing CMM-forming enzyme. The identification of thestrain M6 was carried out according to the method as described in“BISEIBUTSU-NO-BUNRUI-TO-DOTEI” (Classification and Identification ofMicroorganisms), edited by Takeji Hasegawa, published by JapanScientific Societies Press, Tokyo, Japan (1985).

<A. Morphology>

-   -   (1) Characteristic of cells when incubated at 27° C. in nutrient        agar;        -   Existing usually in a rod or coccus shape of 0.4×1.0 to            0.8×3.0 □m,        -   Exhibiting polymorphism,        -   Possessing motility and asporogenicity, and        -   Gram stain, positive;    -   (2) Characteristic of cells when incubated at 27° C. in EYG        agar;        -   Exhibiting a rod-coccus cycle.

<B. Cultural Property>

-   -   (1) Characteristics of colony formed when incubated at 27° C. in        nutrient agar plate;        -   Shape: Circular colony having a diameter of 1-2 mm after 3            days incubation        -   Rim: Entire        -   Projection: Hemispherical shape        -   Gloss: Dull        -   Surface: Smooth        -   Color: Opaque and pale yellow    -   (2) Characteristics of colony formed when incubated at 27° C. in        nutrient agar slant;        -   Growth: Medium        -   Shape: Thread-like    -   (3) Characteristics of colony formed when stub cultured at        27° C. in bouillon and gelatin;        -   Not liquefying bouillon and gelatin.

<C. Physiological Properties>

-   -   (1) VP-test: Negative    -   (2) Indole formation: Negative    -   (3) Hydrolysis of starch: Positive    -   (5) Formation of pigment: Forming no soluble pigment    -   (6) Urease: Negative    -   (7) Oxidase: Positive    -   (8) Catalase: Positive    -   (9) Growth conditions: Growing at a pH of 5.5-10.0 and a        temperature of 15-37° C.    -   (10) Oxygen requirements: Aerobic    -   (11) Major diamino acid of cell wall: Lysine    -   (12) Peptideglycan type of cell wall: Lysine-Alanine    -   (13) N-Acyl type of cell wall: Acetyl    -   (14) Component sugar of cell wall: Galactose, Glucose and        Rhamnose    -   (15) Vitamin requirement: Negative    -   (16) Mol % of guanine (G) plus cytosine (C) of DNA: 70%, and    -   (17) DNA-DNA homology: Having a DNA-DNA homology of 69.3%        between Arthrobacter globiformis ATCC8010

The bacteriological properties were compared with those of knownmicroorganisms with reference to Bergey's Manual of SystematicBacteriology, Vol. 2 (1986). As a result, it was revealed that themicroorganism was identified as of Artherobacter globiformis. Based onthese results, the present inventors named this microorganism“Arthrobacter globiformis M6” and deposited it on Aug. 6, 2003, inInternational Patent Organism, National Institute of Advanced IndustrialScience and Technology, AIST Tsukuba Central 6, 1-1, Higashi 1-ChomeTsukuba-shi, Ibaraki-ken, Japan, and accepted under the accession numberof FERM BP-8448.

In addition to the above-mentioned microorganism and its mutant, amicroorganism capable of producing CMM-forming enzyme of the presentinvention includes other microorganisms such as recombinantmicroorganisms and their mutants, capable of producing CMM-formingenzyme.

The term “the DNA of the present invention” means any DNA encoding theabove-mentioned CMM-forming enzyme. The DNA of the present inventionincludes a DNA originated from the nature and that synthesizedartificially as far as the DNA encodes CMM-forming enzyme.Microorganisms of the genus Arthrobacter, including Arthrobacterglobiformis M6 (FERMBP-8448) can be used as the natural sources of theenzyme. A genomic DNA containing the DNA of the present invention can beobtained from the cells of these microorganisms. Specifically, a genomicDNA containing the DNA can be released extracellularly by the steps ofinoculating any of the microorganisms into a nutrient medium, culturingabout one to three days under aerobic conditions, collecting the cellsfrom the culture, treating the cells with cell-lytic enzymes such aslysozyme and β-glucanase or with ultrasonication. In addition to themethods described above, use of protein-hydrolyzing enzymes such asproteinases, detergents such as SDS and freeze-thaw method are alsoapplicable. The objective DNA can be obtained from the treated cells byusing conventional methods in the art, for example, such asphenol-extraction, alcohol-precipitation, centrifugation andribonuclease-treatment. To artificially synthesize the DNA of thepresent invention, it can be chemically synthesized based on the aminoacid sequence of SEQ ID NO:2. PCR-Method is also applicable to obtainthe DNA by using a genomic DNA containing the DNA as a template and anappropriate chemically synthetic DNA as a primer.

The DNA of the present invention has, usually, a prescribed nucleotidesequence, for example, a nucleotide sequence of SEQ ID NO:3 or anucleotide sequence homologous to SEQ ID NO:3. A variant DNA, having ahomologous nucleotide sequence to SEQ ID NO:3, means that having anucleotide sequence where one or more nucleotides of SEQ ID NO:3 aredeleted, replaced or added without altering the activity of the enzymeencoded thereby. The homology of nucleotide sequence to SEQ ID NO:3 ofsuch a variant DNA is preferable to be, usually, 60% or higher,desirably, 70% or higher, more desirably, 80% or higher, most desirably,90% or higher. The DNA of the present invention encompasses a DNA havinga nucleotide sequence where one or more nucleotides of SEQ ID NO:3 arereplaced with other nucleotides without altering the encoded amino acidsequence based on the degeneracy of genetic code.

The DNA of the present invention can be advantageously used forconstructing a recombinant DNA by inserting to an appropriateself-replicable vector. Recombinant DNAs are usually constructed by aDNA and a self-replicable vector, and they can be relatively easilyprepared by conventional recombinant DNA techniques if the DNA isobtained. Such vectors include, for example, plasmid vectors such aspBR322, pUC18, Bluescript II SK(+), pUB110, pTZ4, pC194, pHV14, TRp7,YEp7 and pBS7; and phage vectors such as λgt·λC, λgt·B, ρ11, φ1 andφ105. To express the DNA of the present invention in E. coli, pBR322,pUC18, Bluescript II SK(+), λgt·λC and λgt·λB can be preferably used.While, to express the DNA of the present invention in Bacillus subtilis,pUB110, pTZ4, pC194, ρ11, φ1 and φ105 can be preferably used. Plasmids,pHV14, TRp7, YEp7 and pBS7 are useful in the case of replicating therecombinant DNA in two or more kinds of hosts. In order to insert a DNAinto these vectors, conventional methods in the art can be used.Specifically, a DNA is inserted into a vector by the steps of cleaving agenomic DNA containing the objective DNA and a self-replicable vector byrestriction enzyme and/or ultrasonication, and ligating the resultingDNA fragment and the resulting vector fragment. The ligation of the DNAfragment and the vector fragment is easily carried out by using a typeII-restriction enzymes, particularly, such as Sau 3AI, Eco RI, Hin dIII,Bam HI, Sal I, Xba I, Sac I and Pst I. The desired recombinant DNA isobtainable by ligating them in vivo or in vitro using a DNA ligase,optionally, after annealing the both fragments. The recombinant DNA thusobtained is unlimitedly replicable by the steps of introducing into anappropriate host and culturing the resulting transformant.

The recombinant DNA thus obtained can be introduced into an appropriatehost-microorganism such as E. coli, B. subtilis, Actinomyces and yeasts.The desired transformant can be obtained by applying thecolony-hybridization method or by selecting a transformant by the stepsof culturing a transformant in nutrient media containing α-1,4 glucanhaving a glucose polymerization degree of 3 or higher, and selecting aclone which produces CMM from saccharides.

Any nutrient culture medium can be used for cultivating a microorganism,including a transformant, capable of producing CMM-forming enzyme of thepresent invention as long as these microorganisms can grow therein andproduce CMM-forming enzyme: For example, synthetic- and natural-culturemedia can be used as nutrient culture media. Any carbon source can beused as long as it is utilized by the microorganisms: Examples of suchcarbon source are saccharides such as starch and phytoglycogen,obtainable from plants; glycogen and pullulan, obtainable from animalsand microorganisms; those hydrolyzates, glucose, fructose, lactose,sucrose, mannitol, sorbitol, and saccharide syrups; and organic acidssuch as citric acid and succinic acid. The concentrations of thesecarbon sources in nutrient culture media are appropriately chosen. Thenitrogen sources usable in the present invention are, for example,inorganic nitrogen compounds such as ammonium salts and nitrates;organic nitrogen compounds such as urea, corn steep liquor, casein,peptone, yeast extract and beef extract. The inorganic ingredientsusable in the invention are, for example, calcium salts, magnesiumsalts, potassium salts, sodium salts, phosphates, manganese salts, zincsalts, iron salts, copper salts, molybdenium salts, and cobalt salts. Ifnecessary, amino acids and vitamins can be suitably used.

The microorganisms of the present invention are cultured under aerobicconditions, usually, at a temperature in the range of 15-37° C. and at apH in the range of 5.5-10, preferably, at a temperature in the range of20-34° C. and at a pH in the range of 5.5-8.5. The cultivation time isset to a time longer than that required for the growth of themicroorganisms, preferably, 10-150 hours. The concentration of dissolvedoxygen is not specifically restricted, but usually 0.5-20 ppm. Theconcentration of dissolved oxygen can be kept within the above range bycontrolling aeration and agitation. The cultivation can be carried outbatch-wise or in a continuous manner.

After culturing the microorganisms capable of producing CMM-formingenzyme according to the method described above, the culture containingthe enzyme of the present invention is recovered. In the case ofculturing a microorganism, Arthrobacter globiformis M6 (FERM BP-8448),the major activity of CMM-forming enzyme is found in the cell-freesupernatant. Both the cell-free supernatant and the culture can be usedas a crude enzyme. Conventional liquid-solid separation methods can beemployed to remove cells from the culture. For example, methods todirectly centrifuge the resultant culture, as well as those to filtratethe culture with pre-coated filters or to separate cells by membranefiltration using plane filter or follow fibers, can be suitably used.While cell-free supernatants thus obtained can be used intact as a crudeenzyme solution, they may be concentrated prior to use. Theconcentration methods usable in the invention are, for example, saltingout using ammonium sulfate, sedimentation using acetone or alcohol, andconcentration using membranes such as plane filters and follow fibers.

CMM-forming enzyme can be subjected to conventional immobilization usingcell-free supernatants and their concentrates. Examples for suchconventional methods are conjugation methods using ion exchangers,covalent bindings and adsorptions using resins and membranes, andinclusion methods using high molecular weight substances.

As described above, a crude enzyme solution can be used intact afterconcentrating it as CMM-forming enzyme of the present invention.Further, CMM-forming enzyme can be advantageously used after separatingor purifying the crude enzyme solution by suitable conventional methodsused in the art. For example, a purified CMM-forming enzyme preparationexhibiting an electrophoretically single band can be obtained bydialyzing a crude enzyme preparation which had been salting out acell-free supernatant or disrupted cells with ammonium sulfate andconcentrating the resultant; and successively purifying the dialyzedsolution on anion-exchange column chromatography using “DEAE-TOYOPEARL650S”; hydrophobic chromatography using “PHENYL-TOYOPEARL 65CM”.

In the case of producing CMM-forming enzyme as a recombinant enzyme, theenzyme may be accumulated intracellularly, depending on the kinds ofhost microorganisms. In such cases, while the cell or the culture can beused intact, the recombinant enzyme can be advantageously used afterextracting it from cells by using osmotic-shock methods or detergents orby disrupting cells using ultrasonication methods or cell-wall digestingenzymes; and separating it from cell or cell debris.

CMM-forming enzyme of the present invention, thus obtained, acts onα-1,4 glucan having a glucose polymerization degree of 3 or higher andproduces CMM having a structure ofcyclo{>6)-α-D-glucopyranosyl-(1>4)-α-glucopyranosyl-(1>6)-α-glucopyranosyl-(1>4)-α-glucopyranosyl-(1>}.It was revealed that the enzyme produces CMM having a structure ofcyclo{>6)-α-D-lucopyranosyl-1>4)-α-glucopyranosyl-(1>6)-α-glucopyranosyl-(1>4)-α-glucopyranosyl-(1>} by the steps of acting onα-1,4 glucan having a glucose polymerization degree of 3 or higher,transferring □-maltosyl moiety by the intermolecular transglycosylationto form 6-□-maltosyl 0-1,4 glucan where α-maltosyl moiety is bound tohydroxyl group at C-6 position at the non-reducing end glucose of α-1,4glucan, and cyclizing the 6-α-maltosyl α-1,4 glucan to form CMM having astructure ofcyclo{>6)-α-D-glucopyranosyl-(1>4)-α-glucopyranosyl-(1>6)-α-glucopyranosyl-(1>4)-α-glucopyranosyl-(1>}.Particularly, CMM-forming enzyme of the present invention may have thefollowing physicochemical properties:

(1) Molecular Weight

-   -   72,000±20,000 daltons when determined on SDS-PAGE;

(2) Isoelectric Point

-   -   pI 3.6±0.5 on isoelectrofocusing ampholine, a carrier ampholyte;

(3) Optimum Temperature

-   -   50 to 55° C. when reacted at pH 6.0 for 30 min;

(4) Optimum pH

-   -   pH 5.5 to 6.0 when reacted at 40° C. for 30 min;

(5) Thermal Stability

-   -   Stable up to 30° C. when incubated at pH 6.0 for 60 min    -   Stable up to 50° C. in the presence of 1 mM Ca²⁺ ion;

(6) pH Stability

-   -   Stable in a pH range at 5.0 to 9.0 when incubated at 4° C. for        24 hours; and

(7) N-Terminal Amino Acid Sequence

-   -   Having an amino acid sequence of SEQ ID NO:1, i.e.        Asp-Pro-Thr-Thr-Ser

α-1,4 Glucan having a glucose polymerization degree of 3 or higher,which can be used as a substrate of CMM-forming enzyme of the presentinvention, includes starch, amylose, amylopectin, glycogen, and theirpartial hydrolyzates such as amylodextrins, maltodextrins,maltooligosaccharides, obtainable by partially hydrolyzing them withamylases and acids. The partial hydrolyzates obtainable by hydrolyzingstarch, amylose, amylopectin, and glycogen by using amylase such asα-amylase (EC 3.2.1.1), maltotetraose-forming amylase (EC 3.2.1.60), andmaltohexaose-forming amylase (EC 3.2.1.98), described in “Handbook ofAmylases and Related Enzymes” published by Pergamon Press Inc., (Tokyo),1988, can be used as the partial hydrolyzates prepared by hydrolyzingwith amylases. Further, starch-debranching enzymes such as pullulanase(EC 3.2.1 41) and isoamylase (EC 3.2.1.68) can be arbitrarily used forpreparing the partial hydrolyzates.

Both subcelestal starches such as those from corn, wheat, rice, etc.,and subterranean starches such as those from potato, sweet potato,tapioca, etc., can be used as substrates. The substrate can bepreferably used in the form of a solution prepared by gelatinizingand/or liquefying starch. The CMM content in the reaction mixture isincreased with decrease of the degree of partial hydrolysis of starch.Therefore, it is preferable that the DE of the partial starchhydrolyzate is, usually, about 20 or lower, desirably, about 12 orlower, more desirably, about 5 or lower. The CMM content as referred toas in the present specification means the value which is calculated bythe following formula:

CMM content (%)={(Weight of CMM formed)/(Total weight of saccharides inthe reaction mixture)}×100

When CMM-forming enzyme is allowed to act on a substrate, the substrateconcentration is not specifically restricted. For example, the reactionby CMM-forming enzyme of the present invention proceeds to form CMM evenin the case of using a substrate solution with a relatively lowconcentration such as 0.1% (w/v). For industrial production, thesubstrate concentration is preferable to be 1% (w/v) or higher, and CMMcan be advantageously produced under the condition. Also, suspensionswith a high concentration, containing insoluble substrates can be usedas the substrate solutions. The reaction temperature used in the presentenzymatic reaction can be set to a temperature at which the reactionproceeds, i.e. a temperature up to about 60° C., preferably, atemperature in a range of 30 to 50° C. The reaction pH is controlled inthe range of, usually, 5 to 9, preferably, 5 to 7. Since the amount ofenzyme and reaction time are closely related, the conditions areadequately chosen with respect to the progress of the objectiveenzymatic reaction.

CMM of the present invention can be obtained in a high content, about30% or higher from starch or its partial hydrolyzate, about 44% fromamylose, by allowing CMM-forming enzyme of the present invention to acton, for example, 1% (w/v) substrate solution containing starch, itspartial hydrolyzate, or amylose. The mechanism of CMM-formation byCMM-forming enzyme is estimated as follows:

(1) The enzyme acts on α-1,4 glucan having a glucose polymerizationdegree of 3 or higher as the substrates and forms 6-α-maltosyl-α-1,4glucan whose glucose polymerization degree is increased by two, having6-α-maltosyl moiety at the non-reducing end, and α-1,4 glucan whoseglucose polymerization degree is decreased by two by catalyzing anintermolecular 6-α-maltosyl transferring reaction to transfer a maltosylmoiety at the non-reducing end of the substrate to hydroxyl group at theC-6 position of the non-reducing end glucose of another α-1,4 glucanmolecule.

(2) The enzyme further acts on 6-α-maltosyl-α-1,4 glucan and forms CMMhaving a structure ofcyclo{>6)-α-D-glucopyranosyl-(1>4)-α-D-glucopyranosyl-(1>6)-α-D-glucopyranosyl-(1>4)-α-D-glucopyranosyl-(1>}and maltooligosaccharide whose glucose polymerization degree isdecreased by four, by catalyzing an intramolecularα-maltosyl-transferring reaction to cyclize and to form CMM.

(3) CMM is formed from α-1,4 glucan newly formed in the above 1) and 2),by the reaction described in the above 1) and 2).

In the above CMM-forming reaction, CMM content in the reaction mixturecan be advantageously improved by using other enzymes together withCMM-forming enzyme. For example, CMM content in the reaction mixture canbe advantageously improved by allowing CMM-forming enzyme together withstarch-debranching enzymes such as isoamylase to act on starch.

The reaction mixture, thus obtained by the above reaction, can be usedintact as a saccharide solution comprising CMM. Optionally, thesaccharide solution comprising CMM can be used after hydrolyzingconcomitant oligosaccharides by allowing one or more enzymes selectedfrom the group consisting of α-amylase, β-amylase, glucoamylase, andα-glucosidase to act on the solution. Usually, a saccharide solutioncomprising CMM is used after purification. Conventional methods used forpurifying saccharides can be arbitrarily selected as the purificationmethod. For example, one or more purification methods selected from thegroup consisting of decoloring with an activated charcoal; desaltingwith ion exchange resins in H- and OH-form; fractionation by columnchromatography such as ion exchange column chromatography, charcoalcolumn chromatography, and silica gel column chromatography; separationusing organic solvents such as alcohol and acetone; separation using amembrane having a suitable separability; fermentation usingmicroorganisms, which utilize and decompose concomitant saccharides butdoes not utilize CMM, such as yeasts; and eliminating the remainingreducing sugar with alkaline treatments; can be arbitrarily used.

More particularly, ion exchange column chromatography can be suitablyused as an industrial-scale preparation of the objective saccharides.The objective CMM or a saccharide composition comprising the same withan improved purity can be advantageously prepared by, for example,column chromatography using a strongly acidic cation exchange resin asdescribed in Japanese Patent Kokai Nos. 23,799/83 and 72,598/83 toremove concomitant saccharides. In this case, any one of fixed bed,moving bed, and semi-moving bed methods can be employed.

A solution comprising CMM, thus obtained, or a saccharide solution withan improved purity of CMM contains CMM in an amount of, usually, 10%(w/w) or higher, desirably, 40% (w/w) or higher, on a dry solid basis,and is usually concentrated to make into a product in a syrupy form. Thesyrupy product can be arbitrarily dried to make into a powdery product.

In order to prepare crystalline CMM, an about 5 to 90% (w/w) solutioncomprising CMM with a purity of about 50% or higher is placed in acrystallizer, and gradually cooled while stirring in the presence of 0.1to 20% (w/w) seed crystal at a temperature of 95° C. or lower,preferably, 10 to 90° C., to obtain a massecuite containing crystallineCMM. Centrifugation can be employed to produce crystalline CMM from themassecuite. Conventional methods such as block pulverization,fluidized-bed granulation, and spray-drying can be employed to preparecrystalline saccharides containing the mother liquor from themassecuite. The resulting crystalline CMM or crystalline saccharidescontaining the mother liquor obtained according to the present inventionis a non-reducing white powder with a mild and relatively low sweetnessand relatively higher stability. Because of this, these saccharides canbe mixed and processed with other materials, especially, amino acids andamino acid-containing substances such as oligopeptides and proteinswithout fear of causing browning, smell and deterioration of othermaterials.

Further, CMM of the present invention has clathrating ability andprevents the volatilization and deterioration of clathrated flavors andeffective ingredients. Therefore, CMM can be used for stabilizing andkeeping flavors and effective ingredients. In this case, the stabilizingeffect by clathrating using CMM can be advantageously enhanced by usingCMM together with other cyclic saccharides such as cyclodectrins,branched cyclodextrins, a cyclic tetrasaccharide having a structure ofcyclo{>6)-α-D-glucopyranosyl-(1>3)-α-D-lucopyranosyl-1>6)-α-D-glucopyranosyl-(1>3)-α-D-glucopyranosyl-(1>} which is disclosed in International PatentPublication Nos. WO 01/90338 A1, WO 02/055708 A1, and WO 02/40659 A1 bythe same applicant as the present invention, branched cyclictetrasaccharides, cyclodextrans, cyclofructans, etc. The cyclicsaccharides such as cyclodextrins are not restricted to products withhigh purities. For example, a starch hydrolyzate comprising variouscyclic saccharides together with a large amount of maltodextrins can beadvantageously used as cyclic saccharides with low purities.

In addition, CMM of the present invention is not substantiallyhydrolyzed by amylase and α-glucosidase. Therefore, CMM is not digestedand adsorbed when orally taken, and is hardly fermented by intestinalbacteria. Since CMM has a remarkably low calorie, it can be used as asubstance like a water-soluble dietary fiber. In the case of using CMMof the present invention as a powdery product, it shows a relatively lowhygroscopicity and has a low adhesiveness and solidification. Theproduct can be used for preventing adhesion and solidification ofpowdery products produced by mixing it with other powders. Intact CMM isa novel sweetener with no toxicity and no harm.

A crystalline CMM can be advantageously used for tablets andsugar-coated tablets in combination with binders such as pullulan,hydroxyethyl starch, and polyvinylpyrrolidone because CMM per se is astable sweetener. Furthermore, CMM of the present invention hasproperties of osmosis-controlling ability, filler-imparting ability,gloss-imparting ability, moisture-retaining ability, viscosity,crystallization-preventing ability for other sugars, less-fermentableproperty, etc.

Thus, CMM and the saccharide compositions comprising the same of thepresent invention can be advantageously used as a sweetener,taste-improving agent, quality-improving agent, stabilizer,color-deterioration preventing agent, excipient, etc., for variouscompositions such as foods and beverages, favorite products, feeds,baits, cosmetics, pharmaceuticals, etc.

CMM of the present invention and the saccharide compositions comprisingthe same can be used intact as a seasoning for sweetening products. Ifnecessary, they can be advantageously used in combination with othersweeteners, for example, powdery syrup, glucose, isomerized sugar,sucrose, maltose, trehalose, honey, maple sugar, sorbitol, maltitol,dihydrochalcone, stevioside, α-glycosyl stevioside, sweetener ofMomordica grosvenori, glycyrrhizin, thaumatin, sucralose, L-aspartylL-phenylalanine methyl ester, saccharine, glycine and alanine; andfillers such as dextrin, starch, and lactose.

Further, powdery products of CMM of the present invention and thesaccharide compositions comprising the same can be arbitrarily usedintact or, if necessary, after mixing with fillers, excipients, binders,etc., and then shaped into various shapes such as granules, spheres,sticks, plates, cubes, and tablets.

CMM of the present invention and the saccharide compositions comprisingthe same have sweetness which well harmonize with other materials havingsour-, salty-, astringent-, delicious-, and bitter-taste; and have ahigh acid- and heat-tolerance. Thus, they can be advantageously used tosweeten and/or improve the taste and quality of general food products.

CMM of the present invention and the saccharide compositions comprisingthe same can be advantageously used as a sweetener, taste-improvingagent, and quality-improving agent for various seasonings such as a soysauce, powdered soy sauce, miso, “funmatsu-miso” (a powdered miso),“moromi” (a refined sake), “hishio” (a refined soy sauce), “furikake” (aseasoned fishmeal), mayonnaise, dressing, vinegar, “sanbai-zu” (a sauceof sugar, soy sauce and vinegar), “funmatsu-sushi-zu” (powdered vinegarfor sushi), “chuka-no-moto” (an instant mix for Chinese dish),“tentsuyu” (a sauce for Japanese deep fat fried food), “mentsuyu” (asauce for Japanese vermicelli), sauce, catsup, “yakiniku-no-tare” (asauce for Japanese grilled meat), curry roux, instant stew mix, instantsoup mix, “dashi-no-moto” (an instant stock mix), mixed seasoning,“mirin” (a sweet sake), “shin-mirin” (a synthetic mirin), table sugar,and coffee sugar. Also, CMM and the saccharide compositions comprisingthe same can be advantageously used to sweeten and to improve the tasteand quality of various “wagashi” (Japanese cakes) such as “senbei” (arice cracker), “arare” (a rice cake cube), “okoshi” (a millet and ricecake), “gyuhi” (a starch paste), “mochi” (a rise paste) and the like,“manju” (a bun with a bean-jam), “uiro” (a sweet rice jelly), “an” (abean-jam) and the like, “yokan” (a sweet jelly of beans), “mizu-yokan”(a soft azuki-bean jelly), “kingyoku” (a kind of yokan), jelly, pao deCastella, and “amedama” (a Japanese toffee); Western confectioneriessuch as a bun, biscuit, cracker, cookie, pie, pudding, butter cream,custard cream, cream puff, waffle, sponge cake, doughnut, chocolate,chewing gum, caramel, nougat, and candy; frozen desserts such as an icecream and sherbet; syrups such as a “kajitsu-no-syrup-zuke” (a preservedfruit) and “korimitsu” (a sugar syrup for shaved ice); pastes such as aflour paste, peanut paste, and fruit paste; processed fruits andvegetables such as a jam, marmalade, “syrup-zuke” (fruit pickles), and“toka” (conserves); pickles and pickled products such as a“fukujin-zuke” (red colored radish pickles), “bettara-zuke” (a kind ofwhole fresh radish pickles), “senmai-zuke” (a kind of sliced freshradish pickles), and “rakkyo-zuke” (pickled shallots); premix forpickles and pickled products such as a “takuan-zuke-no-moto” (a premixfor pickled radish), and “hakusai-zuke-no-moto” (a premix for freshwhite rape pickles) meat products such as a ham and sausage; products offish meat such as a fish ham, fish sausage, “kamaboko” (a steamed fishpaste), “chikuwa” (a kind of fish paste), and “tenpura” (a Japanesedeep-fat fried fish paste); “chinmi” (relish) such as a“uni-no-shiokara” (salted guts of urchin), “ika-no-shiokara” (saltedguts of squid), “su-konbu” (processed tangle), “saki-surume” (driedsquid strips), “fugu-no-mirin-boshi” (a dried mirin-seasoned swellfish),seasoned fish flour such as of Pacific cod, sea bream, shrimp, etc.;“tsukudani” (foods boiled down in soy sauce) such as those of layer,edible wild plants, dried squid, small fish, and shellfish; daily dishessuch as a “nimame” (cooked beans), potato salad, and “konbu-maki” (atangle roll); milk products; canned and bottled products such as thoseof meat, fish meat, fruit, and vegetable; alcoholic beverages such as asynthetic sake, fermented liquor, sake, fruit liquor, low-malt beer andbeer; soft drinks such as a coffee, cocoa, juice, carbonated beverage,sour milk beverage, and beverage containing a lactic acid bacterium;instant food products such as instant pudding mix, instant hot cake mix,instant juice, instant coffee, “sokuseki-shiruko” (an instant mix ofazuki-bean soup with rice cake), and instant soup mix; and other foodsand beverages such as solid foods for babies, foods for therapy, drinks,peptide foods, and frozen foods.

CMM and the saccharide compositions comprising the same can bearbitrarily used to improve the taste preference of feeds and pet foodsfor animals and pets such as domestic animals, poultry, honey bees, silkwarms, and fishes; and also they can be advantageously used as asweetener and taste-improving agent, taste-curing agent,quality-improving agent, and stabilizer for various compositionsincluding favorite products, cosmetics, and pharmaceuticals in a pasteor liquid form such as tobacco, cigarette, toothpaste, lipstick, rouge,lip cream, internal liquid medicine, tablet, troche, cod-liver oil inthe form of drop, oral refrigerant, cachou, and gargle.

When used as a quality-improving agent or stabilizer, CMM and thesaccharide compositions comprising the same can be advantageously usedin biologically active substances susceptible to lose their effectiveingredients and activities, as well as in health foods, functionalfoods, and pharmaceuticals containing the biologically activesubstances. Example of such biologically active substances are liquidpreparations containing lymphokines such as α-, β-, and γ-interferons,tumor necrosis factor-α (TNF-α), tumor necrosis factor-β (TNF-β),macropharge migration inhibitory factor, colony-stimulating factor,transfer factor, and interleukin 2; liquid preparations containinghormones such as insulin, growth hormone, prolactin, erythropoietin, andfollicle-stimulating hormone; liquid biological preparations such as BCGvaccine, Japanese encephalitis vaccine, measles vaccine, live poliovaccine, small pox vaccine, tetanus toxoid, Trimeresurus antitoxin, andhuman immunoglobulin; liquid preparations containing antibiotics such aspenicillin, erythromycin, chloramphenicol, tetracycline, streptomycin,and kanamycin sulfate; liquid preparations containing vitamins such asthiamin, riboflavin, L-ascorbic acid, cod liver oil, carotenoid,ergosterol, tocopherol; highly unsaturated fatty acids and theirderivatives such as EPA, DHA and arachidonic acid; solution of enzymessuch as lipase, esterase, urokinase, protease, β-amylase, isoamylase,glucanase, and lactase; extracts such as ginseng extract, turtleextract, chlorella extract, aloe extract and propolis extract;biologically active substances such as living microorganisms paste ofvirus, lactic acid bacteria, and yeast, and royal jelly. By using CMMand the saccharide compositions comprising the same, the abovebiologically active substances can be arbitrary prepared in healthfoods, functional foods, and pharmaceuticals in a liquid, paste, orsolid form, which have a satisfactorily-high stability and quality withless fear of losing or inactivating their effective ingredients andactivities.

The methods for incorporating CMM or the saccharide compositioncomprising the same into the aforesaid compositions are those which canincorporate CMM and the saccharide compositions into the compositionsbefore completion of their processing, and which can be appropriatelyselected from the following conventional methods; mixing, kneading,dissolving, melting, soaking, penetrating, dispersing, applying,coating, spraying, injecting, crystallizing, and solidifying. The amountof CMM or the saccharide compositions comprising the same to bepreferably incorporated into the final compositions is usually in anamount of 0.1% or higher, desirably, 1% or higher.

The following experiments explain the present invention in detail.

EXPERIMENT 1 Preparation of a Non-Reducing Saccharide

A liquid culture medium consisting of 1.5% (w/v) of “PINE-DEX #4”, apartial starch hydrolyzate commercialized by Matsutani ChemicalIndustries Co., Ltd., Hyogo, Japan, 0.5% (w/v) of “POLYPEPTONE”, a yeastextract commercialized by Nihon Pharmaceutical Co., Ltd., Tokyo, Japan,0.1% (w/v) of “YEAST EXTRACT S”, a yeast extract commercialized by NihonPharmaceutical Co., Ltd., Tokyo, Japan, 0.1% (w/v) of dipotassiumphosphate, 0.06% (w/v) of sodium phosphate dodeca-hydrate, 0.05% (w/v)of magnesium sulfate hepta-hydrate, 0.3% (w/v) of calcium carbonate, andwater was placed in twelve 500 ml-Erlenmeyer flasks in a respectiveamount of 100 ml, sterilized by autoclaving at 121° C. for 20 min, andNov. 16, 2007 cooled. Successively, the culture medium was inoculatedwith Arthrobacter globiformis M6, FERM BP-8448, and followed thecultivation under rotary-shaking conditions at 27° C. and 230 rpm for120 hours. After completion of the culture, about 1.1 L of the culturesupernatant was obtained by centrifuging the culture broth to removecells. One liter of the resulting culture supernatant was used as anenzyme preparation and admixed with one liter of 50 mM acetate buffercontaining 2% (w/v) of soluble starch and 2 mM of calcium chloride andfollowed by the reaction at 40° C. for 24 hours. The reaction wasstopped by heating at about 100° C. for 10 min.

To examine the saccharides in the resulting reaction mixture,saccharides were separated by silica gel thin-layer chromatography(hereinafter, simply abbreviated as “TLC”) using “KIESELGEL 60”, a TLCaluminum plate (10×20 cm) and a solvent (n-butanol/pyridine/water,volume ratio of 6:4:1) and two-times ascending method. The separatedsaccharide on the plate were detected by visualizing the spots withsulfate-methanol method, and glucose with a Rg value of 1.00, maltosewith a Rg value of 0.82, and two kinds of unknown saccharides with Rgvalues of about 0.44 and about 0.21 were detected. It was consideredthat the two kinds of unknown saccharides were formed from solublestarch by the action of an enzyme in the culture supernatant obtainedfrom Arthrobacter globiformis M6. The above “Rg value” means a rate ofsolute migration distance to glucose migration distance on TLC and it iscalculated by the following equation:

Rg value=(solute migration distance/glucose migration distance)

Successively, the above reaction mixture was adjusted to pH 5.0 usinghydrochloric acid, then admixed with 4,000 units/g-dry solid of“TRANSGLUCOSIDASE-LAMANO”, α-glucosidase commercialized Amano EnzymeInc., Aichi, Japan, and 250 units/g-dry solid of glucoamylasecommercialized by Nagase ChemteX Corporation, Osaka, Japan, and followedby the reaction at 50° C. for 16 hours. After completion of thereaction, the reaction was stopped by heating at about 100° C. for 10min. The resulting reaction mixture was subjected to TLC analysis toreveal the saccharides in the mixture. As a result, glucose and thesaccharide showing Rg value of about 0.44 were detected but maltose andthe saccharide showing Rg value of about 0.21 were not. This resultrevealed that maltose and the saccharide showing Rg value of about 0.21are hydrolyzed into glucose by α-glucosidase and glucoamylase but thesaccharide showing Rg value of about 0.44 is not.

Successively, the pH of the reaction mixture was adjusted to 12 byadding sodium hydroxide, and the resulting mixture was incubated at 98°C. for one hour to decompose reducing sugars. After removing insolublesubstances by filtrating the reaction mixture, the resulting filtratewas decolored and desalted using “DIAION SK-1B”, an ion exchange resincommercialized by Mitsubishi Chemical Corporation, Tokyo, Japan, and“IRA 411”, an anion exchange resin commercialized by Organo Corporation,Tokyo, Japan. The resulting solution was filtrated, concentrated usingan evaporator, and dried in vacuo to obtain about 4.0 g of a powderysaccharide, on a dry solid basis.

The saccharide, thus obtained, was subjected to high performance liquidchromatography (abbreviated as “HPLC”, hereinafter) to analyze thesaccharide composition. As shown in FIG. 1, a peak was detected at aretention time of 10.61 min, revealing that the purity of the saccharideis extremely high, i.e., 97% or higher. HPLC was carried out under thefollowing conditions:

-   -   Column: “Shodex SUGAR KS-801”, produced by Showa Denko K. K.,        Tokyo, Japan    -   Eluent: Water    -   Column temperature: 60° C.    -   Flow rate: 0.5 ml/min    -   Detector: “RI-8012”, a refractive index detector produced by        Tosoh Corporation, Tokyo, Japan.

The reducing power of the saccharide was measured by the Somogyi-Nelsonmethod, revealing that it was less than the measurable limit. It wasconcluded that the saccharide was a non-reducing saccharidesubstantially.

EXPERIMENT 2 Structural Analyses of the Non-Reducing SaccharideExperiment 2-1 Mass Spectrometry

The mass of the non-reducing saccharide obtained by the method inExperiment 1 was analyzed using “LCQ Advantage”, a mass spectrometercommercialized by Theremo Electron K.K., Kanagawa, Japan. A sodium-addedmolecular ion with a mass of 671 was remarkably detected and the datarevealed that the mass of the non-reducing saccharide of the presentinvention was 648.

Experiment 2-2 Analysis of Component Sugar

According to conventional method, the component sugar of thenon-reducing saccharide obtained by the method in Experiment 1 wasexamined by hydrolyzing the saccharide to monosaccharide with dilutedsulfuric acid and analyzing the resulting hydrolyzate by using gaschromatography. Only D-glucose was detected in the hydrolyzate,revealing that the saccharide was constructed with D-glucose.Considering with the above mass, it was revealed that the non-reducingsaccharide of the present invention was a cyclic saccharide composed offour D-glucose molecules.

Experiment 2-3 Methylation Analysis

According to conventional method, the non-reducing saccharide obtainedby the method in Experiment 1 was subjected to methylation analysis, andthe resulting methylated products were analyzed by gas chromatography.The result is in Table 1.

TABLE 1 Methylation product Ratio 2,3,4-Trimethylated product 1.032,3,6-Trimethylated product 1.00

As is evident from the result in Table 1, 2,3,4-trimethylated productand 2,3,6-trimethylated product were detected in equimolar amount.Therefore, it was revealed that the non-reducing saccharide, constructedby four D-glucose molecules, of the present invention was constructed bytwo D-glucose molecules whose hydroroxyl groups at C-1 and C-6 positionswere bound with other D-glucose molecule viaglucosidic linkages and twoD-glucose molecules whose hydroxyl groups at C-1 and C-4 positions werebound with other D-glucose molecules via glucosidic linkages.

Experiment 2-4 Nuclear Magnetic Resonance (NMR) Analysis

According to conventional method, the non-reducing saccharide wassubjected to NMR analysis. Its ¹H-NMR spectrum and ¹³C-NMR spectrum arein FIGS. 2 and 3, respectively. Although the results in Experiments 2-1and 2-2 indicated the non-reducing saccharide to be constructed by fourglucose molecules, only 12 signals of carbon atom were detected in its¹³C-NMR spectrum. From the result, it was revealed that the non-reducingsaccharide was a cyclic tetrasaccharide having a symmetrical structure.Two signals, at about 4.93 ppm and about 5.26 ppm, in ¹H-NMR spectrumwere assigned to proton at C-1 position of D-glucose residue, and thesespin-spin coupling constants were about 3.309 Hz (signal at about 4.93ppm) and about 3.677 Hz (signal at about 5.26 ppm), respectively. Fromthe results, it was revealed that both anomer types of hydroxyl groupsat C-1 position of D-glucose residue bound via the 1,4-glucosidic andthe 1,6-glucosidic linkages were α-type.

From the above results, it was revealed that the non-reducing saccharideof the present invention is cyclic maltosylmaltose shown in FIG. 4, i.e.a cyclic tetrasaccharide having a structure ofcyclo{>6)-α-D-glucopyranosyl-(1>4)-α-D-glucopyranosyl-(1>6)-α-D-glucopyranosyl-(1>4)-α-D-glucopyranosyl-(1>}.Since the saccharide, having the above structure, has been unknownbefore, CMM of the present invention is a novel cyclic saccharide.

EXPERIMENT 3 Preparation of CMM-Forming Enzyme

The liquid culture medium, described in Experiment 1, was placed in two500 ml-Erlenmeyer flasks in a respective amount of 100 ml, sterilized byautoclaving at 121° C. for 20 min, cooled and inoculated withArthrobacter globiformis M6, FERM BP-8448, and followed by culturingunder rotary-shaking conditions at 27° C. and 230 rpm for 48 hours toprepare a seed culture.

About 20 L of a fresh preparation of the same liquid culture medium asused in the above seed culture were placed in a 30-L fermenter,sterilized by heating, and then cooled to 27° C. and inoculated withabout 200 ml of the seed culture, followed by the cultivation at 27° C.and pH 5.5 to 8.0 for 96 hours under aeration-agitation conditions.After completion of the cultivation, the resulting culture broth wasdistilled from the fermenter and removed cells by centrifuging at 8,000rpm for 20 min, and about 18 L of culture supernatant was obtained.CMM-forming enzyme activities in the culture broth and culturesupernatant were assayed. About 0.028 unit/ml and about 0.026 unit/ml ofthe enzyme activities were detected in the culture broth and the culturesupernatant, respectively. It was revealed that major part ofCMM-forming enzyme of the present invention, produced by Arthrobacterglobiformis M6, was secreted extracellularly.

EXPERIMENT 4 Purification of CMM-Forming Enzyme

About 9.2 L (Total activity: about 240 units) of the culture supernatantobtained in Experiment 3 was salted out by adding ammonium sulfate togive finally 60% saturation and allowing it to stand at 4° C. for 24hours. The resultant precipitates were collected by centrifuging at11,000 rpm for 30 min, dissolved in 10 mM Tris-HCl buffer (pH 7.5), anddialyzed against the same buffer to obtain about 240 ml of a crudeenzyme solution. The crude enzyme solution had about 0.83 unit/ml (Totalactivity: about 200 units) of CMM-forming enzyme. The crude enzymesolution was subjected to anion-exchange column chromatography using 100ml of “DEAE-TOYOPEARL 650S” gel, an anion-exchange gel commercialized byTosoh Corporation, Tokyo, Japan. CMM-forming enzyme activity wasadsorbed on “DEAE-TOYOPEARL 650S” gel pre-equilibrated with 10 mMTris-HCl buffer (pH 7.5) and when eluted with a linear gradientincreasing from 0 M to 0.4 M of sodium chloride, CMM-forming enzymeactivity was eluted at about 0.22 M of sodium chloride. The activefractions were collected and added ammonium sulfate to give a finalconcentration of 1 M, and then allowed to stand at 4° C. for 24 hours.The enzyme solution was centrifuged to remove precipitates, andsubjected to hydrophobic column chromatography using 10 ml of“PHENYL-TOYOPEARL 650M” gel, a gel commercialized by Tosoh Corporation,Tokyo, Japan. CMM-forming enzyme activity was adsorbed on“PHENYL-TOYOPEARL 650M” gel pre-equilibrated with 20 mM acetate buffer(pH 6.0) containing 1 M of ammonium sulfate and when eluted with alinear gradient decreasing from 1 M to 0 M of ammonium sulfate,CMM-forming enzyme activity was eluted at about 0.1 M of ammoniumsulfate. The amount of enzyme activity, specific activity and yield ofCMM-forming enzyme in each purification step are in Table 2.

TABLE 2 Enzyme* Specific activity activity of enzyme* Yield Purificationstep (units) (units/mg-protein) (%) Culture supernatant 240 0.13 100Dialyzed solution after 200 0.66 63 salting out with ammonium sulfateEluate from ion-exchange 140 7.3 58 column chromatography Eluate fromhydrophobic 96 10 40 column chromatography *CMM-forming enzyme Thefinally purified enzyme preparation of CMM-forming enzyme was assayedfor purify on gel electrophoresis using a 5-20% (w/v) gradientpolyacrylamide gel and detected on the gel as a single protein band,i.e. a high purity preparation.

EXPERIMENT 5 Properties of CMM-Forming Enzyme Experiment 5-1 MolecularWeight

The purified enzyme preparation of CMM-forming enzyme, obtained by themethod in Experiment 4, was subjected to SDS-PAGE and molecular weightof CMM-forming enzyme was measured comparing with molecular weightmarkers, commercialized by Bio-Rad Japan, Tokyo, Japan. It was revealedthat CMM-forming enzyme of the present invention has a molecular weightof 72,000±20,000 daltons.

Experiment 5-2 Isoelectric Point

The purified enzyme preparation of CMM-forming enzyme, obtained by themethod in Experiment 4, was subjected to polyacrylamide gelisoelectrofocusing containing 2% (w/v) “AMPHOLINE”, a carrier ampholytecommercialized by Amersham Biosciences, Tokyo, Japan and isoelectricpoint of CMM-forming enzyme was measured compare with isoelectric pointmarkers, commercialized by Amersham Biosciences, Tokyo, Japan. It wasrevealed that CMM-forming enzyme of the present invention has anisoelectric point (pI) of 3.6±0.5.

Experiment 5-3 Optimum Temperature and Optimum pH of the Enzyme Reaction

Effects of temperature or pH on the enzyme activity were investigatedusing the purified enzyme preparation of CMM-forming enzyme, obtained bythe method in Experiment 4, by varying temperature and pH at the assayof the enzyme. The results are in FIG. 5 (Optimum temperature) and inFIG. 6 (Optimum pH), respectively. It was revealed that the optimumtemperature of CMM-forming enzyme of the present invention was 50 to 55°C. when reacted at pH 6.0 for 30 min and the optimum pH was 5.5 to 6.5when reacted at 40° C. for 30 min.

Experiment 5-4 Thermal Stability and pH Stability of the Enzyme

Thermal stability and pH stability of the enzyme were investigated usingthe purified enzyme preparation of CMM-forming enzyme, obtained by themethod in Experiment 4. Thermal stability of the enzyme was determinedby the steps of incubating enzyme solution (10 mM acetate buffer, pH6.0)under various temperatures for 60 min in the absence or presence of 1 mMCaCl₂, cooling in water, and measuring the residual enzyme activity. pHStability of the enzyme was determined by the steps of incubating enzymesolution in 100 mM buffer at various pHs, and at 4° C. for 24 hours,adjusting the pH to 6.0, and measuring the residual enzyme activity. Theresults are in FIG. 7 (Thermal stability) and in FIG. 8 (pH Stability),respectively. As is evident from the results in FIG. 7, CMM-formingenzyme of the present invention is stable up to 30° C. in the absence ofCaCl₂, and to 50° C. in the presence of 1 mM CaCl₂. It was revealed thatthe thermal stability of the enzyme was improved in the presence of Ca²⁺ion. As is evident from the results in FIG. 8, it was revealed thatCMM-forming enzyme of the present invention was stable in the range ofpH 5.0 to 9.0.

Experiment 5-5 Effects of Metal Ions on the Enzyme Activity

Effects of metal ions on the enzyme activity were investigated using thepurified enzyme preparation of CMM-forming enzyme, obtained by themethod in Experiment 4, in the presence of 1 mM of various metal ionsaccording to the assay method. The results are in Table 3.

TABLE 3 Relative Relative Metal salt activity (%) Metal salt activity(%) None 100 NiCl₂ 90 MgCl₂ 98 CuCl₂ 1 AlCl₃ 13 ZnCl₂ 73 CaCl₂ 99 SrCl₂90 MnCl₂ 97 BaCl₂ 90 FeCl₂ 95 HgCl₂ 2 FeCl₃ 32 PbCl₂ 36 CoCl₂ 95 EDTA 25

As is evident from the results in Table 3, it was revealed thatCMM-forming enzyme activity was remarkably inhibited by Cu²⁺ and Hg²⁺,and also inhibited by Al³⁺, Fe³⁺ and Pb²⁺. Further, it was revealed thatthe enzyme activity was also inhibited by EDTA, a chelating agent formetal ions.

Experiment 5-6 N-Terminal Amino Acid Sequence

N-terminal amino acid sequence of the enzyme was determined using thepurified enzyme preparation of CMM-forming enzyme, obtained by themethod in Experiment 4, by “Model 429HT”, a protein sequencercommercialized by Applied Biosystems Japan, Tokyo, Japan. As a result,it was revealed that the enzyme had the N-terminal amino acid sequenceof SEQ ID NO:1, i.e., Asp-Pro-Thr-Thr-Ser.

Experiment 5-7 Partial Amino Acid Sequence

A part of the purified enzyme preparation of CMM-forming enzyme,obtained by the method in Experiment 4, was dialyzed against 10 mMTris-HCl buffer (pH 9.0) at 4° C. for 18 hours, and the dialyzedsolution was diluted with a fresh preparation of the same buffer to givea concentration of about one mg/ml. One milliliter of the dilutedsolution as was admixed with 20 μg of “LYSYL ENDOPEPTIDASE”commercialized by Wako Pure Chemicals, Ltd, Tokyo, Japan, and incubatedat 30° C. for 16 hours to hydrolyze the enzyme protein. The resultinghydrolyzate was injected to “μ-BONDAPAK C18 column”, having a diameterof 3.9 mm and a length of 150 mm, a HPLC column commercialized by WatersChromatography Div., MILLIPORE Corp., Milford, USA, pre-equilibratedwith 0.1% (v/v) trifluoroacetate containing 8% (v/v) acetonitrile, at aflow rate of 0.9 ml/min and at ambient temperature, and peptides werefractionated using a linear gradient of acetonitrile increasing from 8%(v/v) to 40% (v/v) in 0.1% (v/v) trifluoroacetate over 120 min. Peptidefragments eluted from the column were detected by monitoring theabsorbance at a wavelength of 210 nm. Six kinds of peptide fragmentseluted at a retention time of about 12 min, about 18 min, about 20 min,about 36 min, about 39 min and about 66 min were separately collectedand amino acid sequences of these were analyzed according to the methodin Experiment 5-6. These peptide fragments had amino acid sequences ofSEQ ID NO:4 to 9.

EXPERIMENT 6 Cloning of a DNA Encoding CMM-Forming Enzyme andPreparation of a Recombinant DNA Comprising the DNA and a Transformant

A DNA encoding CMM-forming enzyme was cloned from Arthrobacterglobiformis M6 (FERM BP-8448), and a self-replicable recombinant DNAcontaining the DNA was constructed. Successively, the nucleotidesequence of the DNA encoding the enzyme was determined and atransformant was also prepared.

Experiment 6-1 Preparation of Chromosomal DNA

A liquid culture medium consisting of 2.0% (w/v) of “PINE-DEX #4”, apartial starch hydrolyzate commercialized by Matsutani ChemicalIndustries Co., Ltd., Hyogo, Japan, 1.0% (w/v) of “ASAHIMEAST”, a yeastextract commercialized by Asahi Food & Healthcare Ltd., Tokyo, Japan,0.1% (w/v) of dipotassium phosphate, 0.06% (w/v) of sodium phosphatedodeca-hydrate, 0.05% (w/v) of magnesium sulfate hepta-hydrate, andwater was placed in 500 ml-Erlenmeyer flasks in a respective amount of100 ml, sterilized by autoclaving at 121° C. for 20 min, cooled andinoculated with Arthrobacter globiformis M6, FERM BP-8448, followed bythe cultivation under rotary-shaking conditions at 27° C. and 230 rpmfor 24 hours.

The cells collected from the culture by centrifugation were suspended inTES buffer (pH 8.0), the suspended solution was admixed with lysozyme togive a concentration of 0.05% (w/v), and incubated at 37° C. for 30 min.After freezing the lysate at −80° C. for one hour, the lysate was addedwith TSS buffer (pH 9.0) and heated to 60° C. The solution was addedwith a mixture of TES buffer and phenol, and was vigorously shaken forfive minutes in an ice bath, and the supernatant was collected bycentrifugation. The supernatant was added twice volume of cold ethanol,and resulting precipitate was collected as a crude chromosomal DNA. Thecrude chromosomal DNA was dissolved in SSC buffer (pH 7.1), and admixedwith 7.5 □g of ribonuclease and 125 □g of proteinase, and incubated at37° C. for one hour. The chromosomal DNA was extracted from the reactantby adding chloroform/isoamylalcohol mixture, then, admixed with coldethanol, and the resulting precipitate containing chromosomal DNA wascollected. The purified chromosomal DNA, obtained according to themethod described above, was dissolved in SSC buffer (pH 7.1) to give aconcentration of about one mg/ml and frozen at −80° C.

Experiment 6-2 Preparation of a Recombinant DNA, pBMB1 and aTransformant, BMB1

One-tenth milliliter of purified chromosomal DNA solution, prepared inExperiment 6-1, was admixed with about 100 units of a restrictionenzyme, Bam HI, and incubated at 37° C. for one hour to digest thechromosomal DNA. The resulting DNA fragments corresponding to about3,000 to 6,000 base pairs were collected by agarose gel electrophoresis.A plasmid vector, “BLUESCRIPT II SK(+)®”, commercialized by StratageneCloning System, was completely digested with a restriction enzyme, BamHI by conventional method. A recombinant DNA was obtained by ligating0.5 μg of the digested plasmid vector with about 5 μg of the DNAfragments prepared before by using a “DNA LIGATION KIT”, commercializedby Takara Shuzo Co., Ltd., according to the method described in adocument attached with the kit. Then, a gene library was prepared bytransforming 100 μg portion of the competent cell, “EPICURIAN COLIXL2-BLUE”, commercialized by Stratagene Cloning System, with therecombinant DNA by conventional competent cell method. Thetransformants, thus obtained as gene library, were inoculated into afresh agar plate medium (pH 7.0) containing 10 g/L of tryptone, 5 g/L ofyeast extract, 5 g/L of sodium chloride, 100 mg/L of ampicillin sodiumsalt, and 50 mg/L of 5-bromo-4-chloro-3-indolyl-β-galactoside, andincubated at 37° C. for 24 hours. About four thousand white coloniesgrown on the plate were transferred to and fixed on a nylon membrane,“HYBOND-N+”, commercialized by Amasham Bioscience K.K. Anoligonucleotide having a nucleotide sequence of“5′-GACGTSGTSCCSAACCACACSGCSGACTAC-3′” was chemically synthesized on thebasis of an amino acid sequence of the fourth to 13th of SEQ ID NO:7,which disclosed by the method in Experiment 5-7. A synthetic DNA(probe 1) was obtained by labeling the oligonucleotide with radioisotopeusing [□-³²P] ATP and T4 polynucleotide kinase according to conventionalmethod. Colonies showing remarkable hybridization with probe 1 wereselected from the colonies fixed on a nylon membrane obtained beforeusing conventional colony hybridization, and then five transformantswere obtained.

Successively, an oligonucleotide having a nucleotide sequence of“5′-GACTGGGTSGACATGGGSTTCGACGGSATC-3′” was chemically synthesized on thebasis of an amino acid sequence of first to 10th of SEQ ID NO:8, andlabeled with radioisotope by the same manner described above to makeinto a synthetic DNA (probe 2). The recombinant DNAs were collected fromthese five transformants by conventional method. A recombinant DNAshowing remarkable hybridization with probe 2 was selected from the fiverecombinant DNAs using conventional southern hybridization. Atransformant containing the selected recombinant DNA was named “BMB1”.

According to conventional method, the transformant, BMB1 was inoculatedinto L-broth medium (pH 7.0) containing 100 μg/ml of ampicillin sodiumsalt, and cultured under rotary-shaking conditions at 37° C. for 24hours. After completion of the culture, cells were collected bycentrifugation from the culture, and the recombinant DNA was extractedfrom the cells by conventional alkaline-SDS method. When the nucleotidesequence of the recombinant DNA was analyzed by conventional dideoxymethod, it was revealed that the recombinant DNA contained a DNA havingthe nucleotide sequence of SEQ ID NO:10, 4,467 base pairs, whichoriginated from Arthrobacter globiformis M6 (FERM BP-8448). As shown inFIG. 9, in the recombinant DNA, the DNA was ligated at downstream ofrecognition site of a restriction enzyme, Bam HI. The amino acidsequence deduced from the nucleotide sequence is as shown in parallel inSEQ ID NO:10. The amino acid sequence was compared with amino acidsequences of CMM-forming enzyme of the present invention, i.e., theN-terminal amino acid sequence of SEQ ID NO:1 disclosed by the method inExperiment 5-6 and the internal partial amino acid sequences of SEQ IDNO:4 to 9 disclose by the method in Experiment 5-7. An amino acidsequence of SEQ ID NO:1 was completely identical with that of 41st to45th of the amino acid sequence shown in parallel in SEQ ID NO:10. Aminoacid sequences of SEQ ID NO:4, 5, 6, 7, 8, and 9 were completelyidentical with those of 418th to 426th, 405th to 417th, 323rd to 332nd,164th to 190th, 241st to 265th, and 333rd to 362nd of the amino acidsequence shown in parallel in SEQ ID NO:10, respectively. These resultsindicate that CMM-forming enzyme of the present invention contains theamino acid sequence of SEQ ID NO:2, and that the enzyme is encoded bythe DNA having the nucleotide sequence of SEQ ID NO:3 in the case ofArthrobacter globiformis M6 (FERM BP-8448). An amino acid sequence ofthe first to 40th of that shown in parallel in SEQ ID NO:10 was presumedto be a secretion signal sequence of the enzyme. According to theresults described above, it was revealed that the precursor of theenzyme before secretion had the amino acid sequence shown in parallel inSEQ ID NO:10, and the amino acid sequence was encoded by the nucleotidesequence of SEQ ID NO:10. The recombinant DNA prepared and confirmed thenucleotide sequence as described above was named “pBMB1”.

EXPERIMENT 7 Production of CMM-Forming Enzyme by the Transformant

A liquid culture medium consisting of 5 g/L of “PINE-DEX #4”, a partialstarch hydrolyzate commercialized by Matsutani Chemical Industries Co.,Ltd., Hyogo, Japan, 20 g/L of polypeptone, 20 g/L of yeast extract, 1g/L of sodium phosphate dodeca-hydrate, and water was placed in a 500ml-Erlenmeyer flask in a amount of 100 ml, sterilized by autoclaving at121° C. for 20 min, and cooled. Then, the liquid medium was sterilelyset to pH 7.0, and sterilely admixed with 10 mg of ampicillin sodiumsalt. A transformant, BMB1, obtained by the method in Experiment 6-2,was inoculated into the above liquid medium, and cultured at 27° C. for48 hours under aeration-agitation conditions. Cells and supernatant wereseparately collected from the culture by conventional centrifugation. Inthe case of the cells, whole-cell extract was prepared by ultrasonicdisruption. The ultrasonic disruption was carried out by suspendingcells in 10 mM sodium phosphate buffer (pH 7.0) and disrupting cellsuspension in a ice bath using a ultrasonic homogenizer, “Model UH-600”,commercialized by MST Corporation, Aichi, Japan, and the resultinghomogenate was used as a whole-cell extract.

CMM-forming enzyme activities of the culture supernatant and whole-cellextract, prepared as described above, were assayed, and those valueswere expressed in terms of the activities/ml-culture, respectively. As acontrol, CMM-forming enzyme activities of the culture supernatant andwhole-cell extract of E coli XL-Blue, the host, were assayed afterculturing the host and preparing the culture supernatant and whole-cellextract in the same manner. The results are in Table 4.

TABLE 4 CMM-forming enzyme activity (units/ml-broth) Strain Culturesupernatant Whole cell extract BMB1 0.00 0.05 (Present invention) E.coli 0.00 0.00 (Control) Note: BMB1 and E. coli, in the Table, mean atransformant, BMB1, and E. coli XL2-Blue, respectively.

As is evident from the results in Table 4, it was revealed that thetransformant, BMB1 produced CMM-forming enzyme of the present inventionintracellularly. In the case of the host, E. coli XL2-Blue, no enzymeactivity was detected from either of the culture supernatant and thewhole-cell extract.

The whole-cell extract, obtained by the method in Experiment 7, wasfurther purified by salting out, dialysis and successive columnchromatographies on “DEAE-TOYOPEARL 650S” gel and “BUTYL-TOYOPEARL 650M”gel according to the methods in Experiment 4, and the purified enzymepreparation was analyzed according to the methods in Experiment 5. Asthe results, the molecular weight was about 72,000±20,000 daltons bySDS-polyacrylamide gel electrophoresis, the isoelectric point was about3.6±0.5 by polyacrylamide gel isoelectrofocusing, the optimumtemperature of CMM-forming enzyme activity was about 50 to 55° C., theoptimum pH of the enzyme was about 5.5 to 6.5, the thermal stability wasup to 30° C. in the absence of CaCl₂ and up to about 50° C. in thepresence of 1 mM CaCl₂, and the pH stability was in the range of aboutpH 5.0 to about 9.0. These physicochemical properties were substantiallyidentical to those of the enzyme prepared by the method in Experiment 4.The results described above indicate that CMM-forming enzyme of thepresent invention can be advantageously produced by recombinant DNAtechniques and the productivity of the enzyme can be significantlyimproved by recombinant DNA techniques.

EXPERIMENT 8 Action on Various Saccharides

Substrate specificity of CMM-forming enzyme of the present invention wasinvestigated using various saccharides as substrates. Substratesolutions were prepared by dissolving maltose, maltotriose,maltotetraose, maltopentaose, maltohexaose, maltoheptaose, neotrehalose,trehalose, kojibiose, nigerose, Nov. 16, 2007 isomaltose,isomaltotriose, panose, isopanose, maltitol, maltotriitol, α-, β- orγ-cyclodextrin, amylose, soluble starch, glycogen, pullulan or dextraninto water. Each substrate solution was admixed with acetate buffer (pH6.0) and CaCl₂ to give final concentrations of 20 mM and 1 mM,respectively. Then, each resulting substrate solution was furtheradmixed with one unit/9-substrate, on a dry solid basis, of the purifiedpreparation of CMM-forming enzyme, obtained by the method in Experiment4. Substrate concentration was set to 2% (w/v) and followed by theenzyme reaction at 40° C. and pH 6.0 for 24 hours. Action and thespecificity of the enzyme on these saccharides were confirmed byanalyzing the reaction mixture before and after the reaction by TLCdescribed in Experiment 1. The results are in Table 5.

TABLE 5 Substrate Action* Substrate Action* Maltose − Panose −Maltotriose + Isopanose − Maltotetraose +++ Maltitol − Maltopentaose +++Maltotriitol − Maltohexaose +++ α-Cyclodextrin − Maltoheptaose +++β-Cyclodextrin − Neotrehalose − γ-Cyclodextrin − Trehalose − Amylose +++Kojibiose − Soluble starch +++ Nigerose − Glycogen ++ Isomaltose −Pullulan − Isomaltotriose − Dextran − *In comparison with before andafter the reaction, the symbol, “−” means “Not changed”. The symbol, “+”means “Spot of substrate is slightly decreased and other products aredetected”. The symbol, “++” means “Spot of substrate is markedlydecreased and other products are detected”. The symbol, “+++” means“Spot of substrate is virtually disappeared and other products aredetected”.

As is evident from the results in Table 5, CMM-forming enzyme of thepresent invention acts on maltotetraose, maltopentaose, maltohexaose,and maltoheptaose, and slightly on maltotriose among saccharides tested.Further, CMM-forming enzyme of the present invention acts on amylose,starch, and glycogen. From the results, it was revealed that the enzymeacted on α-1,4 glucans having a glucose polymerization degree of 3 orhigher.

EXPERIMENT 9 Action Mechanism Experiment 9-1 Product from Maltotetraoseby the Enzyme Reaction

A substrate solution was prepared by mixing maltotetraose solution,acetate buffer (pH 6.0) and CaCl₂ to give final concentrations of 1%(w/v), 20 mM, and 1 mM, respectively. The substrate solution was admixedwith one unit/g-substrate, on a dry solid basis, of CMM-forming enzyme,obtained by the method in Experiment 4, and followed by the enzymereaction at 40° C. and pH 6.0. Aliquots were sampled from the reactionmixture with time and the reaction was stopped by keeping at 100° C. for10 min. Saccharide compositions of the samples were measured by HPLCmethod. HPLC was carried out under the following conditions:

-   -   Column: “YMC Pack ODS-AQ303”, produced by YMC Corporation,        Tokyo, Japan;    -   Elute: Water;    -   Column temperature: 40° C.;    -   Flow rate: 0.5 ml/min; and    -   Detection: “RID-10A”, a diffractometer produced by Shimadzu        Corporation, Kyoto, Japan.

The results are in Table 6.

TABLE 6 Reaction time Saccharide composition (%) (hours) G₂* CMM** G₄*MM*** G₆* X**** Others 0 0.0 0.0 97.3 0.0 0.0 0.0 2.7 1 9.0 2.6 69.5 0.53.9 11.3 1.8 2 15.6 6.6 51.7 0.9 5.6 14.0 2.6 4 22.8 12.5 35.5 1.8 5.414.7 3.5 8 31.7 21.3 19.1 3.8 4.1 10.8 5.7 16 36.3 25.6 10.9 6.9 2.5 8.26.5 24 38.7 28.6 6.9 9.6 1.2 7.1 6.1 *“G₂”, “G₄” and “G₆” mean maltose,maltotetraose and maltohexaose, respectively. **CMM means cyclicmaltosylmaltose. ***MM means 6²-O-α-maltosylmaltose. ****“X” is revealedto be α-maltosylmaltotetraose (alias 6⁴-α-maltosylmaltotetraose) inExperiment 9-3 described later.

As is evident from the results in Table 6, it was revealed that maltoseand Saccharide X were remarkably formed from substrate, maltotetraose atthe initial stage of the enzyme reaction (one hour). Also, it was foundthat maltohexaose, CMM, and maltosylmaltose (6²-α-maltosylmaltose, Noncyclic) were slightly formed at the stage. According to the progress ofthe reaction, maltose and CMM were remarkably accumulated and the amountof maltosylmaltose was slightly increased. While, it was revealed thatcontents of Saccharide X and maltohexaose were increased to the stage offour hours, but decreased later. According to the results, it wassuggested that CMM-forming enzyme of the present invention acted onmaltotetraose to form mainly maltose and Saccharide X, and acted onSaccharide X to form CMM with the progress of the reaction. Saccgaride Xwas considered to be an intermediate of CMM-forming reaction frommaltotetraose. Since maltosylmaltose and maltohexaose were formedsimultaneously, it was suggested that the enzyme catalyzed atransglycosylation by maltose unit and Saccharide X was a product ofmaltosyl-transferring reaction.

Example 9-2 Isolation of Saccharide X

Isolation of Saccharide X which was considered to be an intermediate ofCMM-forming reaction from maltotetraose was carried out. Two liters of1% (w/v) maltotetraose aqueous solution was admixed with acetate buffer(pH 6.0) and CaCl₂ to give concentrations of 20 mM and 1 mM,respectively. The resulting substrate solution was admixed with oneunit/g-substrate, on a dry solid basis, of the purified preparation ofCMM-forming enzyme, obtained by the method in Experiment 4, and followedby the enzyme reaction at 40° C. and pH 6.0 for 4 hours. The enzymereaction was stopped by keeping the reaction mixture at 100° C. for 10min. After confirming that Saccharide X was not hydrolyzed by β-amylaseby the preliminary test, the pH of the reaction mixture was adjusted to5.5 and the resulting solution was admixed with five units/g-substrate,on a dry solid basis, of β-amylase, produced by Sigma-Aldrich Japan,Tokyo, Japan, and followed by the enzyme reaction at 50° C. for 16 hoursto hydrolyze remaining maltotetraose into maltose. After stopping thereaction by keeping the reaction mixture at 100° C. for 10 min,insoluble substances were removed from the reaction mixture byfiltration. The resulting filtrate was decolored and desalted using“DIAION WA30”, an ion exchange resin commercialized by MitsubishiChemical Corporation, Tokyo, Japan, “DIAION SK-1B”, a cation exchangeresin commercialized by Mitsubishi Chemical Corporation, Tokyo, Japan,and “IRA 411S”, an anion exchange resin commercialized by OrganoCorporation, Tokyo, Japan. The resulting solution was filtrated andconcentrated using an evaporator to make into a material forfractionation. The concentrate was subjected to preparative HPLC using“YMC-Pack ODS-A R355-15S-15 12A”, a column produced by YMC Corporation,Tokyo, Japan, to purify Saccharide X and a preparation of Saccharide Xwith a purity of 99.3% or higher was obtained in a yield of about 6.7%,on a dry solid basis, from the reaction mixture prepared frommaltotetraose.

Experiment 9-3 Structural Analysis of Saccharide X Experiment 9-3-1 Testfor Forming CMM

To 1% (w/v) aqueous solution of the purified preparation of SaccharideX, obtained by the method in Experiment 9-2, acetate buffer (pH 6.0) andCaCl₂ were mixed to give final concentrations of 20 mM and 1 mM,respectively. The resulting substrate solution was admixed with oneunit/g-substrate, on a dry solid basis, of the purified preparation ofCMM-forming enzyme, obtained by the method in Experiment 4, and followedby the enzyme reaction at 40° C. and pH 6.0 for 24 hours. After stoppingthe reaction by keeping the reaction mixture at 100° C. for 10 min,reaction products were analyzed by TLC and HPLC, described inExperiment 1. As a result, it was revealed that maltose and CMM wereformed from Saccharide X as major products. It was confirmed thatSaccharide X was an intermediate of CMM-forming reaction.

Experiment 9-3-2 Mass Spectrometry

The mass of Saccharide X was analyzed by the method in Experiment 2-1using the purified preparation of Saccharide X, obtained by the methodin Experiment 9-2. A sodium-added molecular ion with a mass of 1,013 wasremarkably detected and the data revealed that the mass of Saccharide Xwas 990. The mass indicated that Saccharide X was constructed by sixD-glucose molecules.

Experiment 9-3-3 Hydrolysis by Pullulanase

A digestion test was carried out by allowing pullulanase, commercializedby Hayashibara Biochemical Laboratories Inc., Okayama, Japan, to act ona 1% (w/v) aqueous solution of the purified preparation of Saccharide X,obtained by the method in Experiment 9-2. The substrate solution wasadmixed with one unit/g-substrate, on a dry solid basis, of pullulanaseand followed by the enzyme reaction at 40° C. and pH 6.0 for 24 hours.After stopping the reaction by keeping the reaction mixture at 100° C.for 10 min, the reaction products were analyzed by TLC and HPLC,described in Experiment 1. As a result, maltose and maltotetraose wereformed from Saccharide X. Therefore, it was revealed that Saccharide Xhad a structure of binding maltose molecule and maltotetraose moleculevia the □-1,6 glucosidic linkage.

Experiment 9-3-4 Methylation Analysis

According to conventional method, the preparation of Saccharide X,obtained by the method in Experiment 9-2, was subjected to methylationanalysis, and the resulting methylated products were analyzed by gaschromatography. The result is in Table 7.

TABLE 7 Methylation product Ratio 2,3,4-Trimethylated product 1.002,3,6-Trimethylated product 4.04 2,3,4,6-Tetramethylated product 0.85

As is evident from the result in Table 7, 2,3,4-trimethylated product,2,3,6-trimethylated product, and 2,3,4,6-tetramethylated product weredetected in a ratio of about 1:4:1. Therefore, it was revealed that,among six glucose molecules, one is glucose whose hydroxyl groups at C-1and C-6 positions were involved in glucosidic linkages, four areglucoses whose hydroxyl groups at C-1 and C-4 positions were involved inglucosidic linkages, and one is glucose whose hydroxyl group at C-1position was involved in glucosidic linkage. Further, from the result,it was revealed that the 1,6-glucosidic linkage existed at glucoseresidue at the non-reducing end in Saccharide X having a structure ofbinding maltose and maltotetraose via the □-1,6 glucosidic linkage.

From the results described above, it was revealed that Saccharide X,formed from maltotetraose by CMM-forming enzyme of the presentinvention, was an intermediate of CMM-forming reaction, and was ahexasaccharide having a structure of binding maltose with hydroxyl groupat C-6 position of the non-reducing end glucose residue of maltotetraosevia α-glucosidic linkage, i.e. α-maltosylmaltotetraose(6⁴-α-maltosylmaltotetraose) represented by the structural formula I.

α-D-Glcp-(1→4)-α-D-Glcp-(1→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-D-Glcp  Structuralformula I

From the results described above, the mechanism of CMM-forming reactionby CMM-forming enzyme of the present invention was estimated as follows:

1) The enzyme acts on α-1,4 glucan having a glucose polymerizationdegree of 3 or higher as the substrate and forms6-α-maltosylmaltooligosaccharide whose glucose polymerization degree isincreased by two, having 6-α-maltosyl moiety at the non-reducing end,and maltooligosaccharide whose glucose polymerization degree isdecreased by two by catalyzing an intermolecular 6-α-maltosyltransferring reaction to transfer a maltosyl moiety at the non-reducingend of the substrate to hydroxyl group at the C-6 position of thenon-reducing end glucose of another α-1,4 glucan molecule.

(2) The enzyme further acts on 6-α-maltosylmalto-oligosaccharide andforms CMM having a structure ofcyclo{>6)-α-D-glucopyranosyl-(1>4)-α-D-glucopyranosyl-(1>6)-α-D-glucopyranosyl-(1>4)-α-D-glucopyranosyl-(1>}and maltooligosaccharide whose glucose polymerization degree isdecreased by four, by catalyzing an intermolecular α-maltosytransferring reaction to cyclize to form CMM.

(3) The enzyme slightly catalyzes an intermolecular 4-α-maltosyltransferring reaction and forms maltooligosaccharide whose glucosepolymerization degree is increased by two and maltooligosaccharide whoseglucose polymerization degree is decreased by two from amaltooligosaccharide.

EXPERIMENT 10 Formation of CMM from Various Substrates

Formation of CMM by the action of CMM-forming enzyme of the presentinvention was investigated using various saccharides as substrates.Maltotriose, maltotetraose, maltopentaose, maltohexaose, amylose,soluble starch, “PINEDEX #100”, a partial starch hydrolyzatecommercialized by Matsutani Chemical Industries Co., Ltd., Hyogo, Japan,or glycogen from corn, commercialized by Q.P. Corporation, Tokyo, Japan,was prepared into a solution.

Each solution (concentration: 1.0% (w/v)) was admixed with acetatebuffer (pH 6.0) and CaCl₂ to give final concentrations of 20 mM and 1mM, further admixed with one unit/g-substrate, on a dry solid basis, ofthe purified preparation of CMM-forming enzyme, obtained by the methodin Experiment 4, and followed by the enzyme reaction at 40° C. and pH6.0 for 48 hours. The reaction was stopped by heating the reactionmixture at 100° C. for 10 min. After treating the reaction mixture withα-glucosidase and glucoamylase by the same manner in Experiment 1, theamount of CMM was determined by HPLC and CMM content of the reactionmixture was measured. The results are in Table 8.

TABLE 8 Substrate CMM content (%) Maltotriose 0.6 Maltotetraose 27.3Maltopentaose 24.4 Maltohexaose 41.6 Maltoheptaose 36.6 Amylose 41.8Soluble starch 31.4 Partial starch hydrolyzate 32.6 Glycogen 29.5

As is evident from the results in Table 8, CMM was formed from allsubstrate tested by the action of CMM-forming enzyme. In the case ofusing maltotriose as a substrate, CMM content was low about 0.6%.However, CMM content was the highest to about 42% in the case of usingamylose as a substrate and higher in the case of using maltohexaose andmaltoheptaose in that order. CMM was also formed from soluble starch, apartial starch hydrolyzate, and glycogen in contents of about 30%.

EXPERIMENT 11 Relationship of CMM-Forming Reaction and the ReducingPower of the Reaction Products

An aqueous solution containing 1.0% (w/v) of soluble starch was admixedwith acetate buffer (pH 6.0) and CaCl₂ to give final concentrations of20 mM and 1 mM, respectively. The resulting substrate solution wasadmixed with one unit/g-solid, on a dry solid basis, of the purifiedenzyme preparation of CMM-forming enzyme, obtained by the method inExperiment 4, and followed by the reaction at 40° C. and pH 6.0. Areaction mixture at the zero-time reaction was obtained by the steps ofsampling the aliquot of the reaction mixture just after adding theenzyme, stopping the reaction by heating at about 100° C. for 10 min,and cooling the sample. Successively, aliquots of the reaction mixturewere withdrawn at the reaction time of 1, 2, 3 and 4 hours, and thesamples were immediately stopped the reactions by heating at about 100°C. for 10 min, and cooled to make into reaction mixtures reacted for 1,2, 3, and 4 hours. The amount of reducing sugars and total sugars in theresulting reaction mixtures were measured by the Somogyi-Nelson methodand Anthrone method. Reducing power of the reaction mixture was definedas the ratio of the amount of reducing sugars to the amount of totalsugars and expressed in percentage. Further, the contents of CMM in thereaction mixtures were measured by the steps of treating the reactionmixtures with α-glucosidase and glucoamylase in the same manner inExperiment 1 and measuring the amounts of CMM by HPLC. The results arein Table 9.

TABLE 9 Reducing Reaction time power CMM content (hour) (%) (%) 0 0.3 01 0.3 4.7 2 0.4 8.4 3 0.5 11.2 4 0.5 13.7

As is evident from the results in Table 9, when CMM was formed byallowing CMM-forming enzyme of the present invention to act on solublestarch, it was revealed that the reducing powers of the reaction mixturewere slightly increased by about 0.2% even when the contents of CMM were10% or higher. These results indicate that CMM-forming enzyme of thepresent invention substantially catalyzes transferring and cyclizingreaction and hardly catalyzes hydrolytic reaction. It was also revealedthat products with low reducing power can be obtained by lowering thereducing power of starches or starch hydrolyzates, i.e., the DE(dextrose equivalent) value before the reaction because the reducingpower is hardly increased, when CMM is formed by allowing the enzyme toact on starches or starch hydrolyzates.

EXPERIMENT 12 Effect of the Addition of Isoamylase on the Formation ofCMM

An aqueous solution containing 1% (w/v) of “PINEDEX #100”, a partialstarch hydrolyzate commercialized by Matsutani Chemical Industries Co.,Ltd., Hyogo, Japan, was admixed with acetate buffer (pH 6.0) and CaCl₂to give final concentrations of 20 mM and 1 mM, respectively. Theresulting substrate solution was admixed with one unit/g-substrate, on adry solid basis, of the purified preparation of CMM-forming enzyme,obtained by the method in Experiment 4, and zero, 125, 250, 500, 1,250or 2,500 units/g-substrate, on a dry solid basis, of isoamylasecommercialized by Hayashibara Biochemical Laboratories Inc., Okayama,Japan, and followed by the enzyme reaction at 40° C. and pH 6.0 for 48hours. The reaction was stopped by heating the reaction mixture at 100°C. for 10 min. Successively, after treating the reaction mixture withα-glucosidase and glucoamylase by the same manner in Experiment 1, theamount of CMM was determined by HPLC and CMM content of the reactionmixture was measured. The results are in Table 10.

TABLE 10 Amount of isoamylase CMM content (Units) (%) 0 32.2 125 40.1250 40.1 500 40.9 1250 41.0 2500 41.7

As is evident from the results in Table 10, it was revealed that CMMcontent in the reaction mixture is increased by adding isoamylase.

EXPERIMENT 12 Effect of the DE Value of Liquefied Starch on theFormation of CMM

Cornstarch was prepared into 2% (w/w) suspension and admixed withcalcium carbonate to give a concentration of 0.1% (w/w). After adjustingpH to 6.0, the resulting suspension was further admixed with “THERMAMYL60L”, an α-amylase commercialized by Novozymes Japan, Chiba, Japan, togive a concentration of 0.2, 0.4, 0.6, 1.0, 1.5, or 2.0% (w/w) per gramstarch. These solutions were reacted at 95° C. for 10 min, autoclaved at120° C., and immediately cooled to about 40° C. to obtain six kinds ofliquefied starch solutions with DE values of 3.1 to 20.4, as shown inTable 11. Each liquefied starch solution was adjusted a finalconcentration of 1% (w/w), admixed with one unit/g-solid of the purifiedpreparation of CMM-forming enzyme, obtained by the method in Experiment4, and followed by the reaction at 40° C. and pH 6.0 for 48 hours. Thereaction was stopped by boiling the reaction mixture for 10 min. Tomeasuring the amount of CMM in the boiled reaction mixture, the reactionmixture was admixed with α-glucosidase and glucoamylase with the samemanner in Experiment 1 and followed by the reaction. The CMM content inthe resulting reaction mixture was obtained by measuring the amount ofCMM by HPLC. The results are in Table 11.

TABLE 11 Amount of CMM α-amylase content (w/w %/g-starch) DE (%) 0.2 3.132.6 0.4 4.8 30.3 0.6 7.9 26.2 1.0 12.6 23.1 1.5 17.4 21.2 2.0 20.4 20.9

As is evident from the results in Table 11, CMM formation by CMM-formingenzyme of the present invention was influenced by the DE value ofliquefied starch. It was revealed that CMM content in the reactionmixture was increased by decreasing DE value, in other word, decreasedby increasing DE value. Particularly, it was revealed that DE value ofthe liquefied starch is preferable to, usually, about 20 or lower,desirably, about 8 or lower, more desirably, about 5 or lower.

EXPERIMENT 14 Preparation of Crystalline CMM

Sixteen liters of an aqueous solution, containing 1.25% (w/v) of amylosecommercialized by Hayashibara Biochemical Laboratories Inc., Okayama,Japan, 20 mM of acetate buffer (pH 6.0), and 1 mM of CaCl₂₁ was admixedwith one unit/g-solid of the purified preparation of CMM-forming enzyme,prepared by the method in Experiment 4, and followed by the reaction at40° C. and pH 6.0 for 90 hours. The reaction was stopped by heating thereaction mixture at about 98° C. for 10 min. The resulting reactionmixture was treated with glucoamylase by the method described inExperiment 1, and reducing sugars in the reaction mixture weredecomposed by the alkaline treatment. The resulting solution wasdecolored by filtration, desalted, filtrated, concentrated and dried invacuo to obtain about 80.5 g, on a dry solid basis, of a powdery CMMproduct. The purity of CMM of the product was 98.9% by HPLC analysis.

The powdery CMM, 36 g, on a dry solid basis, was admixed with 144 g ofwater and completely dissolved by heating to about 90° C. Afterpreserving the solution at about 25° C. for two days, crystallinesubstances were formed. The resulting suspension containing crystallinesubstances was filtrated and the crystalline substances on a filter werecollected. Successively, the crystalline substances were washed with asmall amount of water, collected, and dried under the conditions of anambient temperature and a normal pressure to obtain 21.8 g of powderycrystals. The CMM crystal showed an extremely high purity of 99.9% orhigher by HPLC analysis.

The powdery crystalline CMM was subjected to powdery X-ray diffractionanalysis using “RAD-IIX”, an X-ray diffractometer produced by RigakuCorporation, Tokyo, Japan. As shown in FIG. 10, a powdery X-raydiffraction pattern having major diffraction angles (2θ) of 5.6°, 9.3°,16.5°, and 27.1° was obtained. The moisture content of the powderycrystal was measured by Carl-Fisher method, revealing 12.8% (w/W). Fromthe result, it was revealed that the crystal is a hydrous crystal whichhas five molecules of water to one molecule of CMM.

In addition, the powdery crystalline CMM was subjected tothermogravimetric analysis, and then a thermogravimetric curve shown inFIG. 11 was obtained. From the thermogravimetric curve, it was revealedthat the weight corresponding to five molecules of water was decreasedby elevating the temperature to about 100° C. and the weight caused bythe thermal decomposition of intact CMM was decreased at the temperatureof about 280° C. From these results, it was revealed that hydrouscrystalline CMM of the present invention was converted in a normalpressure into anhydrate by releasing five molecules of water per onemolecule of the crystal when the temperature was elevated to 100° C.

EXPERIMENT 15 Concentration of CMM in Saturated Aqueous Solution

To investigate the saturated concentration of CMM to water with atemperature of 25° C., 10 ml of water was put in a glass vessel attachedwith an airtight stopper, and then admixed with an excess amount, whichcan not be dissolved completely, of powdery hydrous crystalline CMM,obtained by the method in Experiment 14. Successively, the glass vesselwas sealed, and the suspension was stirred two days with keeping thetemperature at 25° C. to give a saturated solution. After removing thesolid CMM by filtrating the saturated solution, the moisture content ofthe filtrate was measured by the drying loss method to determine theconcentration of CMM in saturated solution. As a result, the saturatedconcentration of CMM to water with a temperature of 25° C. was revealedto be about 8.0% (w/w).

EXPERIMENT 16 Degree of Sweetness of CMM

A powdery hydrous crystalline CMM, obtained by the method in Experiment14, was dissolved in water to make into a 5% (w/w) aqueous solution, andthe resulting aqueous solution was used for testing the degree ofsweetness. As controls, aqueous solutions containing sucrose(commercially available granulated sugar) was prepared by dissolvingsucrose to give concentrations of 0.5 to 5% (w/w). By the sensoryevaluation carried out by five panelists, the degree of sweetness of CMMwas estimated to be about 20% of that of sucrose. It was revealed thatCMM is a saccharide with a low sweetness.

EXPERIMENT 17 Thermal Stability of CMM

A powdery hydrous crystalline CMM was dissolved in water for preparingan aqueous solution containing 7% (w/v) of CMM. Eight milliliters eachof the resulting solution was pored into a glass tube, sealed closely,and then heated at 120° C. for 30 to 90 min. After cooling the solution,the degree of coloring of the solution was measured. Further, the purityof CMM in the solution was measured by HPLC. The degree of coloring wasdefined as an absorbance at 480 nm using a 1 cm-cell. The results are inTable 12.

TABLE 12 Heating time Degree of coloring Purity (min) (A480 nm) (%) 0 0100 30 0 100 60 0 100 90 0 100

As is evident from the results in Table 12, aqueous solutions of CMMwere not colored, and the purities of CMM were not decreased even in thecase of heating to a high temperature, 120° C. It was revealed that CMMis stable under the heating condition.

EXPERIMENT 18 pH Stability of CMM

The powdery hydrous crystalline CMM, obtained by the method inExperiment 14, was dissolved in various buffers (20 mM) to make intoeight kinds of aqueous solutions containing 4% (w/v) of CMM, adjusted topH 2 to 9, as shown in Table 13. Eight milliliters of each solution wasput in a glass tube, sealed and then heated at 100° C. for 24 hours.After cooling the solution, the degree of coloring and the purity of CMMwere measured in the same manner in Experiment 17. The results are inTable 13.

TABLE 13 Degree of coloring Purity pH Kind of buffer (A480 nm) (%) 2.0Acetate 0 93 3.0 Acetate 0 100 4.0 Acetate 0 100 5.0 Acetate 0 100 6.0Tris-HCl 0 100 7.0 Tris-HCl 0 100 8.0 Tris-HCl 0 100 9.0 Ammonium 0 100

As is evident from the results in Table 13, aqueous solutions of CMMwere not colored in a wide pH range of 2 to 9 even in the case ofheating a high temperature, 100° C. for 24 hours. Although the purity ofCMM was slightly decreased by the decomposition at pH 2, nodecomposition was observed in a pH range of 3 to 9. It was revealed thatCMM is extremely stable even in the case of boiling under wide pHconditions.

EXPERIMENT 19 Amino-Carbonyl Reaction

The powdery hydrous crystalline CMM, obtained by the method inExperiment 14, was dissolved in water. The resulting solution wasadmixed with commercially available super high-grade glycine andphosphate buffer to make into an aqueous solution containing 2.5% (w/v)of CMM, 0.5% (w/v) glycine, and 50 mM phosphate buffer (pH 8.0). As acontrol, an aqueous solution containing maltose and glycine was preparedin the same manner using maltose except for the powdery hydrouscrystalline CMM. Four milliliters each of the solution was put in aglass tube, sealed and then heated at 100° C. for 30, 60, or 90 min.After cooling the solutions, the degrees of coloring of the solutionswere measured to estimate the degree of amino-carbonyl reaction. Thedegree of coloring was defined as an absorbance at 480 nm using a 1cm-cell. The results are in Table 14.

TABLE 14 Degree of coloring (A480 nm) Heating time Maltose (min) CMM(Control) 0 0.00 0.00 30 0.00 0.02 60 0.00 0.08 90 0.00 0.17

As is evident from the results in Table 14, maltose solution, thecontrol of the test, caused browning by heating in the presence ofglycine. While, the solution containing CMM of the present inventionshowed no browning by heating in the presence of glycine. It wasrevealed that CMM is a stable saccharide which hardly causesamino-carbonyl reaction (Maillard reaction).

EXPERIMENT 20 Amino-Carbonyl Reaction

The powdery hydrous crystalline CMM, obtained by the method inExperiment 14, and commercially available polypeptone commercialized byNihon Pharmaceutical Co., Ltd., Tokyo, Japan, were dissolved indeionized water to make into an aqueous solution containing 5% (w/v) ofCMM and 5% (w/v) of polypeptone. As a control, an aqueous solutioncontaining maltose and polypeptone was prepared in the same manner usingmaltose except for the powdery hydrous crystalline CMM. Four milliliterseach of the solution was put in a glass tube, sealed and then heated at120° C. for 30, 60, or 90 min. After cooling the solutions, the degreesof coloring of the solutions were measured to estimate the degree ofamino-carbonyl reaction. As a blank test, a solution containingpolypeptone only was heated in the same manner. The degree of coloringwas defined as an absorbance at 480 nm using a 1 cm-cell aftersubtracting the absorbance of the blank test. The results are in Table15.

TABLE 15 Degree of coloring (A480 nm) Heating time Maltose (min) CMM(Control) 0 0.00 0.00 30 0.00 0.10 60 0.00 0.30 90 0.00 0.62

As is evident from the results in Table 15, maltose solution, thecontrol of the test, caused browning by heating in the presence ofpolypeptone. While, the solution containing CMM of the present inventionshowed no browning by heating in the presence of polypeptone. It wasrevealed that CMM is a stable saccharide which hardly causesamino-carbonyl reaction (Maillard reaction).

EXPERIMENT 21 Clathrating Action of CMM

The powdery hydrous crystalline CMM, obtained by the method inExperiment 14, was dissolved in deionized water to make into an aqueoussolution containing 8% (w/v) of CMM. One hundred grams of the aqueoussolution was admixed with 1.2 g of methanol, 1.7 g of ethanol, or 2.2 gof acetic acid as flavor components for allowing CMM to clathrate theflavor components. Successively, each solution was filtrated, and theresulting filtrate was freeze-dried to remove unclathrated flavorcomponent. As a control, “ISOELITE P”, a mixture of branchedcyclodextrins commercialized by Maruha Corporation, Tokyo, Japan, whichhas been known to have a clathrating activity, was tested by the sameprocedure. In order to measure the amount of flavor component clathratedin the freeze-dried powder, 1 g of each freeze-dried powder wasdissolved in 5 ml of water and admixed with 5 ml of diethylether toextract the flavor component, and the extraction was repeated once more.The amount of the extracted flavor component in diethylether wasmeasured by gas-chromatography. The results are in Table 16.

TABLE 16 Amount of flavor component clathrated (mg/g-freeze- Objectivedried powder) flavor component CMM Branched CD* Methanol 4.30 3.23Ethanol 4.20 8.67 Acetic acid 30.55 38.14 *CD: Cyclodextrin

As is evident from the results in Table 16, it was revealed that CMM hasa clathrating activity. The strength of the activity of CMM was about1.3-folds, 0.5-fold, and 0.8-fold in the cases of methanol, ethanol, andacetic acid, respectively, on a weight basis, in comparison with thoseof branched cyclodextrin.

EXPERIMENT 22 Digestibility of CMM

According to the method of Okada et al., described in Journal ofJapanese Society of Nutrition and Food Sciences, vol. 43, 23-29 (1990),the digestibility of CMM by salivary α-amylase, artificial gastricjuice, pancreas amylase, and small intestinal enzymes were investigatedusing the powdery hydrous crystalline CMM, obtained by the method inExperiment 14. Maltitol, which has been known as a hardly digestivesaccharide, was used as a control. The results are in Table 17.

TABLE 17 Degradation rate (%) Maltitol Digestive enzyme CMM (Control)Salivary α-amylase 0 0 Artificial gastric juice 0 0 Pancreas α-amylase 00 Small intestinal enzymes 0 4

As is evident from the results in Table 17, CMM was not digested at allby either of salivary amylase, artificial gastric juice, pancreasamylase and small intestinal enzymes. It was revealed that CMM is one ofhardly digestive saccharides.

EXPERIMENT 23 Test for Fermentability of CMM

According to the method of Oku et al., described in Journal ofNutritional Science and Vitaminology, vol. 37, 529-544 (1991), thefermentability of CMM by rat cecal contents were investigated using thepowdery hydrous crystalline CMM, obtained by the method in Experiment14. The cecal contents was collected under an anaerobic condition bykilling a Wister-rat under anesthesia and suspended into 4-folds byvolume of 0.1 M sodium bicarbonate aqueous solution, and then used forthe test. CMM was added to the cecal contents by about 7% (w/w) of theweight of the cecal contents. The amounts of CMM at just after mixingand after 12 hours were determined by gas-chromatography. As theresults, the concentrations of CMM in the cecal contents just afteradding and after 12 hours were 68.5 mg/g-cecal contents and 63.0mg/g-cecal contents, respectively. About 92% of CMM was not fermentedand remained. It was revealed that CMM is one of hardly fermentablesaccharides.

EXPERIMENT 24 Acute Toxicity Test

By using mice, CMM obtained by the method in Experiment 14 was orallyadministrated to the mice for its acute toxicity test. As a result, itwas revealed that CMM is a safe substance with a relatively lowtoxicity, and that no mouse died even when administrated with it at thehighest possible dose. Though not so accurate, the value of LD₅₀ of CMMwas 5 g/kg-mouse weight or higher.

From the above results in Experiments 22 to 24, it was revealed that CMMcan be advantageously used for diet sweeteners, excipient forhigh-sweetness sweetener, thickener for diet foods and beverages,fillers, excipients, dietary fibers, materials for substitute of fats,etc., as a non- or low calorie edible material because it is hardlydigested and adsorbed when orally administrated.

The followings explain the process for producing CMM or saccharidecompositions comprising the same in Examples 1 to 6, and compositionscomprising CMM or saccharide compositions comprising the same inExamples 7 to 23.

EXAMPLE 1

According to the method in Experiment 3, Arthrobacter globiformis M6(FERM BP-8448) was cultivated to obtain the seed culture. Successively,a liquid medium containing 3.0% (w/v) of “PINE-DEX #100”, a partialstarch hydrolyzate commercialized by Matsutani Chemical Industry Co.,Ltd., Hyogo, Japan), 3.6% (w/v) of “HI-NEUTE SMS”, soy proteinoligopeptides commercialized by Fuji Oil Co., Ltd., Osaka, Japan, 0.1%(w/v) of dipotassium phosphate, 0.06% (w/v) of sodium phosphatedehydrate, 0.05% (w/v) of magnesium sulfate heptahydrate, 0.3% (w/v) ofcalcium carbonate, and water was placed in a 30-L fermenter in an amountof about 20 L, sterilized by heating, and cooled to 27° C. Then, 1%(v/v) of the liquid medium of the seed culture was inoculated into theliquid medium, and the bacteria was cultured with keeping a temperatureat 27° C. and pH at 5.5 to 8.0 for 96 hours under aeration-agitationconditions. After completion of the cultivation, cells were removed byfiltrating with SF-membrane and about 18 L of the resulting culturefiltrate was collected. Further, the filtrate was concentrated using aUF-membrane and about 1 L of a concentrated enzyme solution, containing3.8 units/ml of CMM-forming enzyme activity, was obtained.

EXAMPLE 2

A potato starch was prepared into a 1% (w/v) starch suspension, admixedwith calcium carbonate to give a final concentration of 1 mM, adjustedto pH 6.0, and then gelatinized by heating at 95° C. for about 20 min.After cooling the resulting substrate solution to about 40° C., theconcentrated enzyme solution containing CMM-forming enzyme, obtained bythe method in Example 1, was admixed with the substrate solution to givea ratio of 0.26 ml (about one unit)/g-dry solid of starch, and followedby the enzymatic reaction at pH 6.0 and 40° C. for 48 hours. Afterkeeping to 95° C. for 30 min, the reaction mixture was cooled andfiltrated. According to conventional manner, the resulting filtrate wasdecolored with activated charcoal, desalted and purified with ionexchangers in H— and OH— forms. Then, the purified solution wasconcentrated to give a concentration of 65% (w/v) and a syrup containingCMM was obtained in a yield of about 90%, on a dry solid basis. Thesyrup contained, on a dry solid basis, 31.4% (w/w) of CMM, 2.2% (w/w) ofmaltose, 1.5% (w/w) of maltotriose, and 65.9% (w/w) of othersaccharides. Since the product has a relatively low reducing power, mildsweetness, adequate viscosity, moisture-retaining ability, andclathrating activity, it can be advantageously used in variouscompositions such as foods and beverages, cosmetics, and pharmaceuticalsas a sweetener, taste-improving agent, flavor-improving agent,quality-improving agent, syneresis-preventing agent, stabilizer,discoloration-preventing agent, excipient, clathrating agent, and basefor powderization.

EXAMPLE 3

A tapioca starch was prepared into a 1% (w/v) starch suspension, admixedwith calcium carbonate to give a concentration of 0.1% (w/v), adjustedto pH 6.5, and admixed with 0.2%/g-starch of “THERMAMYL 60 L”, anα-amylase commercialized by Novo Industries A/S, Copenhagen, Denmark,and then incubated at 95° C. for 10 min. After autoclaving at 120° C.for 20 min, the reaction mixture was cooled rapidly to about 40° C. tomake into a liquefied starch solution with a DE of about 3. Theliquefied starch solution was admixed with 0.26 ml (about oneunit)/g-dry solid starch of the concentrated enzyme solution containingCMM-forming enzyme, obtained by the method in Example 1, and 1,000units/g-dry solid starch of isoamylase commercialized by HayashibaraBiochemical Laboratories Inc., Okayama, Japan, and followed by theenzymatic reaction at pH 6.0 and 40° C. for 48 hours. After heating to95° C. for 30 min, the reaction mixture was cooled and filtrated.According to conventional manner, the resulting filtrate was decoloredwith activated charcoal, desalted and purified with ion exchangers in H—and OH— forms. Then, the purified solution was concentrated to give aconcentration of 60% (w/v) and a syrup containing 41.1% (w/w), on a drysolid basis, of CMM was obtained. The syrup as a saccharide solution wassubjected to a column chromatography using “AMBERLITE CR-1310”(Na-form), a strongly acidic cation-exchange resin commercialized byOrgano Corporation, Tokyo, Japan. The resin was packed into fourjacketed stainless steel columns having a diameter of 5.4 cm, which werethen cascaded in series to give a total gel bed depth of 20 m. Under theconditions of keeping the inner column temperature at 60° C., thesaccharide solution was fed to the columns in a volume of 5% (v/v) andfractionated by feeding to the columns hot water heated to 60° C. at anSV (space velocity) of 0.13 to obtain high CMM content fractions. Whilemonitoring the saccharide composition of elute by HPLC, and then the lowmolecule fractions including the saccharide fraction comprising CMM werecollected and the fractions were purified, concentrated and spray-dried.As a result, a powdery product comprising CMM was obtained in a yield ofabout 54%, on a dry solid basis. The product contained, on a dry solidbasis, 63.2% of CMM, 7.4% of maltose, 6.2% of maltotriose, and 23.2% ofother saccharides. Since the product has a relatively low reducingpower, mild sweetness, adequate viscosity, moisture-retaining ability,and clathrating activity, it can be advantageously used in variouscompositions such as foods and beverages, cosmetics, and pharmaceuticalsas a sweetener, taste-improving agent, flavor-improving agent,quality-improving agent, syneresis-preventing agent, stabilizer,discoloration-preventing agent, excipient, and clathrating agent.

EXAMPLE 4

A corn starch was prepared into a 1% (w/v) starch suspension, admixedwith calcium carbonate to give a concentration of 0.1% (w/v), adjustedto pH 6.0, and admixed with 0.2%/g-starch of “NEOSPITASE”, an α-amylasecommercialized by Nagase ChemteX Corporation, Osaka, Japan, and thenincubated at 85° C. to 95° C. for 20 min. After autoclaving at 120° C.for 20 min, the reaction mixture was cooled rapidly to about 40° C. tomake into a liquefied starch solution with a DE of about 3. Theliquefied starch solution was admixed with 0.26 ml (about oneunit)/g-dry solid starch of the concentrated enzyme solution containingCMM-forming enzyme, obtained by the method in Example 1, and 1,000units/g-dry solid starch of isoamylase commercialized by HayashibaraBiochemical Laboratories Inc., Okayama, Japan, and followed by theenzymatic reaction at pH 6.0 and 40° C. for 48 hours. After heating to95° C. for 30 min, the reaction mixture was cooled to about 50° C. andadjusted to pH 5.0. Then, the reaction mixture was admixed with 100units/g-starch of “GLUCOZYME”, a glucoamylase commercialized by NagaseChemteX Corporation, Osaka, Japan, and followed by the enzyme reactionat pH5.0 and 50° C. for 16 hours. After heating the reaction mixture to95° C. and keeping for 30 min, it was cooled and filtrated. According toconventional manner, the resulting filtrate was decolored with activatedcharcoal, desalted and purified with ion exchangers in H— and OH— forms.Then, the purified solution was concentrated to give a concentration of60% (w/v) and a syrup comprising CMM was obtained in a yield of about95%, on a dry solid basis. The product contained, on a dry solid basis,42.6% (w/w) of CMM, 53.0% (w/w) of glucose, and 4.4% (w/w) of othersaccharides. Since the product has a mild sweetness, adequate viscosity,moisture-retaining ability, and clathrating activity, it can beadvantageously used in various compositions such as foods and beverages,cosmetics, and pharmaceuticals as a sweetener, taste-improving agent,flavor-improving agent, quality-improving agent, syneresis-preventingagent, stabilizer, discoloration-preventing agent, excipient,clathrating agent, and base for powderization.

EXAMPLE 5

According to conventional method, the syrup comprising CMM, obtained bythe method in Example 4, was hydrogenated to convert reducing saccharideinto sugar alcohols. The resulting reaction mixture was purified,concentrated, dried in vacuo, and pulverized and a powdery productcomprising CMM was obtained in a yield of about 90%, on a dry solidbasis. The product contained, on a dry solid basis, 42.6% of CMM, 53.2%of sorbitol, and 4.2% of other sugar alcohols. Since the productsubstantially shows no reducing power, hardly causes amino-carbonylreaction, and has a low reducing power, mild sweetness, adequateviscosity, moisture-retaining ability, and clathrating activity, it canbe advantageously used in various compositions such as foods andbeverages, cosmetics, and pharmaceuticals as a sweetener,taste-improving agent, flavor-improving agent, quality-improving agent,syneresis-preventing agent, stabilizer, discoloration-preventing agent,excipient, and clathrating agent.

EXAMPLE 6

In order to increase the content of CMM, the syrup comprising CMM,obtained by the method in Example 4, was subjected to a columnchromatography using the strongly acidic cation exchange resin in a saltform according to the method in Example 3. CMM high content fractionswere collected and purified, and a CMM high content solution, comprisingCMM in a content of about 90%, on a dry solid basis, was obtained in ayield of about 40%, on a dry solid basis. The solution was concentratedand continuously crystallized to make into a massecuite. The resultingmassecuite was centrifuged using a basket-type centrifugal machine toremove remaining syrup. The resulting crystal was washed with a smallamount of water and dried through a hot air, and hydrous crystalline CMMin a high purity was obtained in a yield of about 25%, on a dry solidbasis. The product is a high purity hydrous crystalline CMM with apurity of 99% or higher. The product shows extremely low reducing powerand hardly causes amino-carbonyl reaction. Further, the product shows nohygroscopicity, and good handling. Since the product has a mildsweetness, adequate viscosity, moisture-retaining ability, clathratingactivity, and low digestibility, it can be advantageously used invarious compositions such as foods and beverages, cosmetics, andpharmaceuticals, and industrial reagents and chemical materials as asweetener, material of low calorie foods, taste-improving agent,flavor-improving agent, quality-improving agent, syneresis-preventingagent, stabilizer, discoloration-preventing agent, excipient,clathrating agent, and base for powderization.

EXAMPLE 7 Sweetener

To 0.8 part by weight of hydrous crystalline CMM, obtained by the methodin Example 6, 0.2 part by weight of “TREHA®”, hydrous crystallinetrehalose commercialized by Hayashibara Shoji Inc., Okayama, Japan, 0.01part by weight of “αG-SWEET”, α-glycosyl-stevioside commercialized byToyo Sugar Refining Co., Ltd, Tokyo, Japan, and 0.01 part by weight of“ASPERTAME”, L-aspartyl-L-phenylalanine-methyl-ester commercialized byAjinomoto Co., Inc., Tokyo, Japan, were mixed to homogeneity andgranulated using a granulator to make into a sweetener in a granuleform. The product has a good sweetness and shows about 2-folds highersweetness than sucrose. The product has substantially no or low caloriebecause CMM is a low-digestive and less fermentable saccharide. Sincethe product is stable with no fear of deterioration under thepreservation at ambient temperature, it can be advantageously used as asweetener with a high quality, low calorie, and low-cariogenicity.

EXAMPLE 8 Hard Candy

Fifty parts by weight of a syrup comprising CMM, obtained by the methodin Example 4, was admixed with 100 parts by weight of sucrose solutionwith a sucrose concentration of 55% (w/v) with heating. Then, themixture was concentrated under a reduced pressure to give a moisturecontent of less than 2%. The resulting concentrate was admixed with 0.6part by weight of citric acid and suitable amounts of lemon flavor andcoloring, shaped into hard candy according to conventional method. Theproduct shows a satisfactory non-adhesion, taste, flavor, and hardlycauses the crystallization of sucrose. The product is a high qualityhard candy with low hygroscopicity and no fluidity.

EXAMPLE 9 Chewing Gum

Three parts by weight of gum base was softened by heating and melting,and then admixed with two parts by weight of anhydrous maltitiol, twoparts by weight of xylitol, two parts by weight of hydrous crystallineCMM obtained by the method in Example 6, one part by weight of hydrouscrystalline trehalose, and suitable amounts of flavor and colorings. Themixture was kneaded by a roll, shaped and packed to make into chewinggum. Since the product has a satisfactory texture, taste, and flavor, itis preferable as a chewing gum with a low-cariogenicity, and lowcalorie.

EXAMPLE 10 Sweetened Condensed Milk

Four parts by weight of a syrup comprising CMM, obtained by the methodin Example 2, and two parts by weight of sucrose were dissolved in 100parts by weight of material milk. The resulting mixture was sterilizedby heating with a plate heater, concentrated to give a concentration of70%, and then packed in a can to make into a product. Since the producthas a mild sweetness and good flavor, it can be advantageously used forseasoning fruits, coffee, cocoa, black tea, and the like.

EXAMPLE 11 Lactic Acid Bacteria Beverage

One hundred seventy-five parts by weight of skim milk, 100 parts byweight of a powdery product comprising CMM, obtained by the method inExample 3, and “NYUKA-OLIGO”, a lactosucrose high content powdercommercialized by Hayashibara Shoji Inc., Okayama, Japan, were dissolvedinto 1,500 parts by weight of water, and then the resulting mixture wassterilized at 65° C. for 30 min. After cooling the mixture to 40° C., 30parts by weight of a lactic acid bacterium was inoculated to the mixtureas a starter according to conventional method, and cultured at 37° C.for eight hours to obtain a lactic acid bacteria beverage. The producthas a satisfactory flavor and keeps the lactic acid bacterium stablybecause it comprises oligosaccharides and CMM. Further, the product ispreferably used as a lactic acid bacteria beverage having agrowth-promoting activity for bifidobacteria and a function-regulatingactivity for intestine.

EXAMPLE 12 Powdery Juice

To 33 parts by weight of a powdery orange juice, produced by aspray-drying method, 50 parts by weight of powdery hydrous crystallineCMM, obtained by the method in Example 6, parts by weight of anhydrouscrystalline maltitol, 0.65 part by weight of anhydrous citric acid, 0.1part by weight of malic acid, 0.2 part by weight of2-O-α-glucosyl-L-ascorbic acid, 0.1 part by weight of sodium citrate,0.5 part by weight of pullulan, and suitable amount of powdery flavorwere mixed with stirring and the resulting powdery mixture waspulverized to make into a fine powdery product. Then, the powderyproduct was subjected to a fluidized bed granulator and its exhausttemperature was set to 40° C. A suitable amount of a syrup comprisingCMM, obtained by the method in Example 2, was sprayed on the powderyproduct and granulated for 30 min and the resulting product was weightedand packed to make into a product. The product is a powdery juice with afruit-juice content of about 30%. Since the product shows no strangetaste and smell, it has a high quality and commercial value as alow-calorie juice.

EXAMPLE 13 Custard Cream

One hundred parts by weight of corn starch, 100 parts by weight of asyrup comprising CMM, obtained by the method in Example 2, 60 parts byweight of hydrous crystalline trehalose, 40 parts by weight of sucrose,and one part by weight of sodium chloride were mixed well, and then 280parts by weight of whole egg was further admixed with the mixture.Successively, 1,000 parts by weight of boiled milk was gradually admixedwith the resulting mixture and the resulting solution was continuouslystirred on an open flame. The heating was stopped at the point that cornstarch was completely gelatinized to give a transparency. After coolingthe mixture, a suitable amount of vanilla essence was admixed with themixture, weighted, and packed to make into a custard cream product. Theproduct is a high quality custard cream with a satisfactory gloss andflavor, whose retrogradation of starch is inhibited.

EXAMPLE 14 Premix for “uiro”

Ninety parts by weight of rice powder, 20 parts by weight corn starch,70 parts by weight of anhydrous crystalline maltitol, 50 parts by weightof a powder comprising CMM, obtained by the method in Example 5, andfour parts by weight of pullulan were mixed to homogeneity to make intoa premix for “uiro” (a Japanese rice cake). The premix for “uiro”, asuitable amount of ground green tea and water was kneaded, put into acontainer, and steamed for 60 min to make into a “uiro” with groundgreen tea. The product has a satisfactory gloss, mouth feel, and flavor.Since the retrogradation of starch of the product is inhibited, theproduct keeps the quality well and is preferable as a “uiro” with a lowcalorie.

EXAMPLE 15 “An” Bean Jam

According to conventional method, 10 parts by weight of “azuki” bean asmaterial and water was mixed and boiled and the tannin, harshness, andwater soluble components were removed, and then about 21 parts by weightof “tsubu-an” of “azuki” bean was obtained. To the row “an”, 14 parts byweight of sucrose, five parts by weight of a syrup comprising CMM,obtained by the method in Example 2, and four parts by weight of waterwere mixed and the resulting mixture was boiled. The mixture was furtheradmixed with a small amount of salad oil and kneaded without crushingthe “tsubu-an” and about 35 parts by weight of “an” product wasobtained. The product has a good stability with no browning andsyneresis and shows a satisfactory mouthfeel and flavor. It can bepreferably used as a material for confectionaries such as “an”-buns,“manju” (a Japanese bean-jam cake), “dango” (a Japanese rice cake),“monaka” (a Japanese bean-jam cake), “hyoka” (ice milk).

EXAMPLE 16 Bread

One hundred parts by weight of wheat flour, two parts by weight ofyeast, five parts by weight of sucrose, one part by weight of a powdercomprising CMM, obtained by the method in Example 3, 0.1 part by weightof mineral food and water was mixed and kneaded according toconventional method. The resulting dough was fermented at 26° C. for twohours, aged for 30 min, and then baked into bread. The product is breadwith a high quality, satisfactory color and texture, adequateelasticity, and mild sweetness.

EXAMPLE 17 Ham

To 1,000 parts by weight of dark meat of pork, 15 parts by weight ofsodium chloride and three parts by weight of potassium nitrate werepenetrated and then preserved for one day in a refrigerated room. Theresulting pork was soaked into a pickled solution composed of 500 partsby weight of water, 100 parts by weight of sodium chloride, three partsby weight of potassium nitrate, 40 parts by weight of a powdercomprising CMM, obtained by the method in Example 5, and spices, forseven days in a refrigerated room. Successively, according toconventional method, the soaked pork was washed with cold water, rolledwith a string, smoked, cooked, cooled and packed to make into a hamproduct. The product is a high-quality ham with a satisfactory color andflavor.

EXAMPLE 18 Powdery Peptide Product

To one part by weight of “HI-NUTE S”, 40% soybean peptides solution forfoods, commercialized by Fuji Oil Co., Ltd., Osaka, Japan, two parts byweight of powdery hydrous crystalline CMM, obtained by the method inExample 6, was mixed and the resulting mixture was put into a plastictray, dried at 50° C. under a reduced pressure, and pulverized to makeinto a powdery peptide product. The product has a satisfactory flavorand is useful as a material for premix, low-calorie confectionaries forice dessert. Further, the product is useful as a less-digestive dietaryfiber and antiflaturent for a fluid diet for oral- or tube-intake.

EXAMPLE 19 Cosmetic Cream

According to conventional method, two parts by weight ofpolyoxiethylenglycol mono-stearate, five parts by weight ofself-emulsified glycerin mono-stearate, two parts by weight of a powderyhydrous crystalline CMM, obtained by the method in Example 6, one partby weight of “αG-RUTIN”, α-glucosyl rutin, commercialized by HayashibaraInc., Okayama, Japan, one part by weight of liquid paraffin, 10 parts byweight of glycerin-trioctanoate and a suitable amount of preservativewere mixed and dissolved by heating. The resulting mixture was furtheradmixed with two parts by weight of lactic acid, five parts by weight of1,3-butylen glycol, and 66 parts by weight of purified water, and theresulting mixture was emulsified using a homogenizer. The homogenizedmixture was further admixed with a suitable amount of flavor and stirredto make into a cosmetic cream. The product has an antioxidative activityand satisfactory stability, and can be advantageously used as a sunburnpreventive, skin-care agent and whitening agent for skin.

EXAMPLE 20 Toothpaste

Forty-five parts by weight of calcium monohydrogen phosphate, 1.5 partsby weight of sodium lauryl sulfate, 25 part by weight of glycerin, 0.5part by weight of polyoxyethylene sorbitanlaurate, 10 parts by weight ofa powder comprising CMM, obtained by the method in Example 5, 0.02 partby weight of saccharin, and 18 parts by weight of water were mixed tomake into a toothpaste. The product is toothpaste whose bad taste isimproved and shows a satisfactory availability without losing thewashing property of surfactant.

EXAMPLE 21 Solid Agent for a Fluid Diet

One hundred parts by weight of a powder comprising CMM, obtained by themethod in Example 3, 200 parts by weight of hydrous crystallinetrehalose, 200 parts by weight of a maltotetraose high content powder,270 parts by weight of powdery egg yolk, 209 parts by weight of skimmilk, 4.4 parts by weight of sodium chloride, 1.8 parts by weight ofpotassium chloride, four parts by weight of magnesium sulfate, 0.01 partby weight of thiamine, 0.1 part by weight of sodium L-ascorbate, 0.6parts by weight of vitamin E acetate, and 0.04 part by weight ofnicotinic acid-amide were mixed to make into a composition. Twenty-fivegrams each of the composition was packed into a damp proof laminatepouch, and the pouch was heat-sealed to make into a product. Since theproduct is enriched with less-digestive dietary fiber by CMM, it can beadvantageously used for supplying energy to living bodies as a fluiddiet to regulate the function of intestine by taking orally or throughtube into nasal cavity, stomach, and intestine.

EXAMPLE 22 Tablet

Fifty parts by weight of aspirin, 14 parts by weight of a powderyhydrous crystalline CMM, obtained by the method in Example 6, and fourparts by weight of corn starch were mixed sufficiently. Then, accordingto conventional method, the mixture was shaped into a tablet with asickness of 5.25 mm and 680 mg/tablet using a tablet machine. Theproduct was prepared by applying the shaping ability of CMM. The productshows no hygroscopicity and has a satisfactory physical strength anddecay property in water.

EXAMPLE 23 Ointment for Curing Wound

One hundred parts by weight of a powdery hydrous crystalline CMM,obtained by the method in Example 6, 300 parts by weight of maltose, and50 parts by weight of a methanol solution containing three parts byweight of iodine, were mixed to make into an ointment for curing woundwith an adequate extendability and adhesive property. Since thevolatilization of iodine and methanol was prevented by CMM, the productis an ointment with a high marketability and less change over time.Since iodine in the product has an antimicrobial activity and maltose inthe product acts as an energy-supplement for cells, the curing period isshortened and wound surface is cured completely.

INDUSTRIAL APPLICABILITY

According to the present invention, a novel cyclic saccharide having astructure ofcyclo{>6)-α-D-lucopyranosyl-1>4)-α-D-glucopyranosyl-(1>6)-α-D-lucopyranosyl-(1>4)-α-D-glucopyranosyl-(1>},which has been unknown, i.e., CMM can be provided in a large amount byproducing the saccharide using CMM-forming enzyme. Since CMM is anon-reducing saccharide, it does not cause browning by reacting withamino compounds through amino-carbonyl reaction (Maillard reaction).Further, since CMM is a cyclic saccharide and has a clathratingactivity, it can be used for inhibiting the volatilization of clathratedcompounds and stabilizing them. The present invention, enabling toprovide a novel cyclic maltosylmaltose, contributes to various fieldssuch as foods and beverages, cosmetics, and pharmaceuticals. The presentinvention, having these outstanding functions and effects, is asignificantly important invention that greatly contributes to this art.

1. A purified cyclic maltosylmaltose-forming enzyme which has anactivity of forming a cyclic maltosylmaltose having a structure ofcyclo{>6)-α-D-glucopyranosyl-(1>4)-α-D-lucopyranosyl-(1>6)-α-D-glucopyranosyl-(1>4)-α-D-glucopyranosyl-(1>}from α-1,4 glucan having a glucose polymerization degree of 3 or higher.2. The purified cyclic maltosylmaltose-forming enzyme of claim 1, whichhas the following physicochemical properties: (1) Molecular weight72,000±20,000 daltons on SDS-PAGE; (2) Isoelectric point pI 3.6±0.5 onisoelecrofocusing a carrier ampholyte; (3) Optimum temperature 50-55° C.when reacted at pH 6.0 for 30 mm; (4) Optimum pH 5.5 to 6.5 when reactedat 40° C. for 30 mm; (5) Thermal stability Stable up to the temperatureof 30° C. when incubated at pH 6.0 for 60 mm; Stable up to thetemperature of 50° C. when incubated at pH 6.0 for 60 mm in the presenceof 1 mM Ca2+ ion; and (6) pH Stability Stable in a range of pH 5.0 to9.0 when incubated at 4° C. for 24 hours.
 3. The purified cyclicmaltosylmaltose-forming enzyme of claim 1, having an amino acid sequenceof SEQ ID NO:1 as N-terminal amino acid sequence.
 4. The purified cyclicmaltosylmaltose-forming enzyme of claim 1, which has an amino acidsequence of SEQ ID NO:2 or an amino acid sequence having deletion,replacement, or addition of one or more amino acid residues of SEQ IDNO:2 without altering the enzyme activity.
 5. The purified cyclicmaltosylmaltose-forming enzyme of claim 1; wherein said α-1,4 glucanhaving a glucose polymerization degree of 3 or higher is one or moresaccharides selected from the group consisting of maltooligosaccharide,maltodextrin, amylodextrin, amylose, amylopectin, soluble starch,liquefied starch, gelatinized starch and glycogen.
 6. The purifiedcyclic maltosylmaltose-forming enzyme of claim 1, which is derived froma microorganism.
 7. The purified cyclic maltosylmaltose-forming enzymeof claim 6, wherein said microorganism belongs to the genusArthrobacter.
 8. The purified cyclic maltosylmaltose-forming enzyme ofclaim 7, wherein said microorganism belonging to the genus Arthrobacteris Arthrobacter globiformis M6 (International Patent OrganismDepository, National Institute of Advanced Industrial Science andTechnology, Accession No. FERN BP-8448) or a mutant thereof.
 9. Aprocess for producing a cyclic maltosylmaltose-forming enzyme,comprising the steps of: culturing a microorganism capable of producingthe cyclic maltosylmaltose-forming enzyme of claim 1 in a nutrientculture medium; and collecting the cyclic maltosylmaltose-forming enzymeof claim 1 from the resulting culture.
 10. The process of claim 9,wherein said microorganism belongs to the genus Arthrobacter.
 11. Theprocess of claim 10, wherein said microorganism belonging to the genusArthrobacter is Arthrobacter globiformis M6 (International PatentOrganism Depository, National Institute of Advanced Industrial Scienceand Technology, Accession No. FERM BP-8448) or a mutant thereof.
 12. Amethod for forming a cyclic maltosylmaltose having a structure ofcyclo{>6)-α-D-glucopyranosyl-(1>4)-α-D-lucopyranosyl(1>6)-α-D-glucopyranosyl-(1>4)-D-glucopyranosyl-(1>},comprising a step of allowing the cyclic maltosylmaltose-forming enzymeof claim 1 to act on a solution containing α-1,4 glucan having a glucosepolymerization degree of 3 or higher.
 13. The method of claim 12,wherein said α-1,4 glucan having a glucose polymerization degree of 3 orhigher is one or more saccharides selected from the group consisting ofmaltooligosaccharide, maltodextrin, amylodextrin, amylose, amylopectin,soluble starch, liquefied starch, gelatinized starch and glycogen.
 14. Aprocess for producing a cyclic maltosylmaltose having a structure ofcyclo{>6)-α-D-glucopyranosyl-(1>4)-α-D-glucopyranosyl-(1>6)-α-D-glucopyranosyl-(1>4)-α-D-glucopyranosyl-(1>},comprising allowing the cyclic maltosylmaltose-forming enzyme of claim 1to act on a solution obtained by gelatinizing and/or liquefying starch.15. The process of claim 14, where the DE value of said solutionobtained by gelatinizing and/or liquefying starch is 20 or lower. 16.The process of claim 14, comprising the steps of: allowing saidcyclicmaltosylmaltose-forming enzyme together with isoamylase to act ona solution obtained by gelatinizing and/or liquefying starch; andoptionally, further allowing one or more enzymes selected from the groupconsisting of (α-amylase, β-amylase, cyclodextrin glucanotransferase,glucoamylase, and α-glucosidase, to act on the solution.
 17. The processof claim 14, comprising the steps of: allowing said cyclicmaltosylmaltose-forming enzyme together with isoamylase to act on asolution obtained by gelatinizing and/or liquefying starch; optionally,further allowing one or more enzymes selected from the group consistingof α-amylase, β-amylase, cyclodextrin glucanotransferase, glucoamylase,and (α-glucosidase, to act on the solution; and purifying the resultantmixture by one or more methods selected from the group consisting offractionation by column chromatography, separation by membrane,fermentation by a microorganism, and elimination by alkaline treatment.18. The process of claim 14, where the product comprises the cyclicmaltosylmaltose in an amount of 1% (w/w) or higher, on a dry solidbasis.
 19. The process of claim 14, where the product is in the form ofa syrup, massecuite, amorphous powder, amorphous solid, crystal, orcrystalline solid.